Study Guide: Hematopoiesis and Immune Cell Development PDF

Study Guide: Key Concepts in Hematopoiesis and Immune Cell Development 1. Hematopoiesis Overview Definition and properties of hematopoietic stem cells (HSCs), including multipotency and self-renewal. Differentiation pathways into common progenitors: ○ Myeloid (macroph...

Study Guide: Key Concepts in Hematopoiesis and Immune Cell Development 1. Hematopoiesis Overview Definition and properties of hematopoietic stem cells (HSCs), including multipotency and self-renewal. Differentiation pathways into common progenitors: ○ Myeloid (macrophages, neutrophils, etc.). ○ Lymphoid (B cells, T cells, NK cells). 2. B Cell Development Development stages: Pro-B → Pre-B → Immature → Mature B cells. Mechanisms of antibody diversity: ○ VDJ recombination and the role of RAG proteins. ○ Junctional diversity via nucleotide additions/deletions. 3. T Cell Development Development stages from Pro-T → Single Positive T cells (CD4/CD8). Diversity generation through recombination and mutations. Differences in alpha and beta chains of the TCR. 4. Proteins and Factors Key transcription factors: RAG-1/2, E47, EZA. Their roles in immune cell maturation. 5. Immunological Mechanisms Testing for autoimmunity. Gene silencing and epigenetic regulation. 6. Experimental Insights Chromatin structure (euchromatin vs. heterochromatin). Results from radiation studies on immune cell recovery. Study Guide: Hematopoiesis, Immune Cell Development, and Related Mechanisms 1. Hematopoiesis Overview Definition: The process of forming blood and immune cells from hematopoietic stem cells (HSCs), occurring in the bone marrow. HSC Properties: ○ Multipotency: Ability to differentiate into all blood cell types. ○ Self-renewal: Maintain their population through replication. Stages and Differentiation Pathways: ○ HSC → Multipotent Progenitor (MPP): MPPs cannot self-renew like HSCs. Differentiate into: Common Myeloid Progenitors (CMP): Generate macrophages, neutrophils, and other innate immune cells. Common Lymphoid Progenitors (CLP): Generate B cells, T cells, and NK cells. Experimental Insight: ○ Radiation Studies: Mice injected with HSCs recover immune and red blood cell populations, showing their regenerative and multipotent abilities. 2. B Cell Development Stages: ○ Pro-B Cells: Rearrangement of heavy chain genes begins. ○ Pre-B Cells: Expression of pre-BCR for antigen recognition starts. ○ Immature B Cells: Express IgM on the surface. ○ Mature B Cells: Express both IgM and IgD as BCRs. Immunoglobulin (Ig) Gene Rearrangement: ○ B cells achieve diversity through VDJ recombination: 1. V (Variable), D (Diversity), and J (Joining) segments rearrange to form the variable region of the Ig heavy chain. 2. Light Chain involves only V and J recombination. ○ Role of RAG Proteins: 1. RAG-1 and RAG-2 initiate recombination by recognizing recombination signal sequences. 2. Essential for creating functional BCRs; their absence leads to immunodeficiency. ○ Junctional Diversity:  1. Addition or deletion of nucleotides by Tdt enzyme enhances variability. ○ Diversity Pairing: 1. Random pairing of heavy and light chains forms unique antigen-binding sites. Antibody Production: ○ B cells differentiate into: 1. Plasma Cells: Secrete antibodies. 2. Memory B Cells: Provide long-term immunity. 3. T Cell Development Location: T cells originate in the bone marrow but mature in the thymus. Stages: ○ Pro-T Cells: Migrate to the thymus. ○ Pre-T Cells: TCR beta chain rearrangement begins. ○ Double Negative (DN): Lack CD4 and CD8 markers; early stages of TCR gene rearrangement. ○ Double Positive (DP): Express both CD4 and CD8; TCR alpha chain rearranges. ○ Single Positive (SP): Commit to either CD4 (helper) or CD8 (cytotoxic) lineage based on TCR interactions. TCR Diversity: ○ Achieved through VDJ recombination: Beta Chain: Involves VDJ recombination. Alpha Chain: Only VJ recombination. ○ Mechanisms of Diversity: Random mutations and overlapping segments. RAG Proteins: Crucial for recombination. Tdt Enzyme: Adds nucleotides to enhance variability. Role of CD4 and CD8: ○ CD4: Helper T cells coordinate immune responses. ○ CD8: Cytotoxic T cells kill infected cells. 4. Immunological Mechanisms Autoimmunity: ○ Immature B cells are tested for self-reactivity. ○ If auto-reactive antibodies are produced, light chain rearrangement can occur to avoid attacking self-antigens. Gene Silencing:  ○ Prevents B cells from becoming T cells via epigenetic mechanisms (e.g., methylation). Mutations: ○ In RAG Proteins: Lead to immunodeficiency (no functional BCRs or TCRs). ○ In TCR or Ig Genes: Result in reduced diversity or self-reactive cells, which are eliminated. 5. Experimental Insights Radiation Studies: ○ Killing immune cells with radiation demonstrates the regenerative role of HSCs when injected back into irradiated mice. Chromatin States: ○ Euchromatin: Active regions where genes are frequently transcribed. ○ Heterochromatin: Silenced regions near the nuclear lamina. ○ B and T cell gene clusters are spatially organized to ensure lineage-specific transcription. Chromosome Isolation: ○ Steps for identifying specific chromosomes: 1. Fix cells with paraformaldehyde. 2. Use detergent to create membrane holes. 3. Stain chromosomes with fluorescent probes matching specific sequences. 6. Key Proteins and Transcription Factors RAG-1/2: ○ Essential for initiating VDJ recombination. ○ Absence leads to lack of functional BCRs and TCRs. E47: ○ A helix-loop-helix protein critical for enhancing B and T cell-specific gene expression. EZA: ○ Regulates transcription during immune cell development. Other Factors: ○ CTCF: Regulates chromatin structure and gene expression. 7. Miscellaneous Cellular Mechanisms  Thymic Changes: ○ Younger mice have larger thymuses, while older mice experience thymic shrinkage. Impact of Recombination Errors: ○ Errors can lead to apoptosis or reduced diversity. Cell Transformation: ○ Tumor cells lose contact inhibition and grow in soft agar, showing malignant properties. 8. Common Questions How do cells react if recombination goes wrong? ○ Apoptosis or reduced immune cell diversity. How is diversity ensured in immune cells? ○ Random combinations of gene segments, mutations, and chromatin organization. Why do B cells not become T cells? ○ Specific gene silencing prevents lineage crossover. What happens in autoimmunity? ○ Mechanisms like light chain rearrangement help avoid self-reactivity. Detailed Study of Chromatin and Transcription Factors Chromatin Structure and Transcription Activity Euchromatin: ○ Loosely packed DNA accessible for transcription. ○ Contains actively transcribed genes, often positioned toward the interior of the nucleus. Heterochromatin: ○ Densely packed DNA that is transcriptionally inactive. ○ Associated with the nuclear lamina and gene silencing. Transcription Factor Families Transcription factors bind to DNA and regulate gene expression, critical for immune cell development: 1. Helix-loop-helix Proteins: ○ E.g., E47, which dimerizes to bind enhancers. ○ Necessary for B and T cell lineage development. 2. Homeodomain Proteins: ○ Bind specific DNA sequences. ○ Regulate genes involved in cellular differentiation. 3. Zinc-finger Proteins: ○ Use zinc ions to stabilize their DNA-binding domains. 4. Leucine Zipper Proteins: ○ Function through dimerization and target specific DNA motifs like TATA sequences. Mechanism for B Cell-Specific Gene Activation E47 Protein: ○ A helix-loop-helix protein essential for regulating genes in both B and T cells. ○ Binds to enhancers and ensures the transcription of critical genes. CTCF: ○ Plays a role in chromatin looping, which regulates the spatial organization of genes. EZA and Pax5: ○ Key regulators in immune cell lineage commitment and chromatin remodeling. 10. Experimental Techniques for Gene Localization  Steps to Visualize Specific DNA Sequences: 1. Cell Fixation: Use paraformaldehyde to preserve cell structure. 2. Membrane Permeabilization: Apply detergents to create holes in the membrane for probe access. 3. DNA Denaturation: Heat or chemically treat DNA to separate strands. 4. Probe Hybridization: Insert fluorescent probes that bind to target DNA sequences. 5. Imaging: Observe under a fluorescence microscope to identify chromosomal loci. 11. Mutations, Tumorigenesis, and Cellular Behavior Contact Inhibition: ○ Normal cells stop growing when they touch neighboring cells. ○ Loss of Contact Inhibition: Leads to uncontrolled cell growth, often seen in cancer. Mutagenic Transformation: ○ Mutations in key genes like Ras (oncogene) promote cancer by enabling cells to grow independently of a solid surface. ○ Tumor cells can form colonies in soft agar, a hallmark of cancerous behavior. Cancer-Related Insights: Oncogenes: ○ Normal genes that, when mutated, drive cancer progression. Metastasis: ○ Ability of tumor cells to invade other tissues, spreading cancer throughout the body. 12. Summary of Key Cellular Mechanisms Primary Cells: ○ Directly isolated from organisms; have limited growth potential. Immortalized Cells: ○ Mutated or transformed to divide indefinitely in culture. Confluency: ○ When cells cover a culture plate fully, stopping division due to space limitation unless mutations override this mechanism. 13. Chromosome Territories and Enhancers Chromosome Territories Definition: Chromosome territories refer to the distinct regions of the nucleus occupied by individual chromosomes. Key Structures: ○ Nuclear Envelope: Surrounds the nucleus. ○ Plasma Membrane: Outer boundary of the cell. ○ Chromosomes remain localized within specific territories to avoid intermingling, allowing precise gene regulation. Regulatory Elements Critical Regulatory Elements: ○ Promoter (P): Located at the transcription start site (TSS); initiates transcription by recruiting RNA polymerase II. ○ Enhancer (E): Enhances the transcription of genes by interacting with promoters, often located far from the TSS. TSS (Transcription Start Site): ○ The point where transcription begins, producing RNA that eventually leaves the nucleus via nuclear pores. Enhancers Function: ○ Activates transcription by serving as a binding site for transcription factors. ○ Without enhancers, transcription may not occur. Testing Enhancer Function: ○ CRISPR Techniques: Used to delete enhancer regions and observe the effect on gene expression. ○ Prove enhancer activity by detecting transcription factor binding at enhancer sites. Characteristics: ○ Enhancers can vary in protein orders or spacing between binding sites. ○ Multiple enhancers work in a coordinated manner to ensure specificity. ○ Enhancer sequences may be conserved across different organisms. Transcription Factor Families 1. Helix-loop-helix Proteins: ○ Bind enhancers to regulate transcription.  2. Homeodomain Proteins: ○ Involved in developmental processes. 3. Zinc-finger Proteins: ○ Bind to DNA using zinc-stabilized domains. 4. Leucine Zipper Proteins: ○ Mediate DNA binding and dimerization. Enhancer Variations and Specificity Combinatorial Complexity: ○ Example: Sequence 1: CAXXTG 899g TATA Sequence 2: CAXXTG 899g CABCG TATA ○ These sequences generate different regulatory outcomes despite sharing similar motifs. Evolutionary Insight: ○ Enhancers may exhibit conserved sequences across certain organisms, supporting evolutionary stability Questions and Short Answers 1. Experiment to Determine if Cells Are Self-Renewing Design: ○ Label hematopoietic stem cells (HSCs) with a fluorescent marker. ○ Transplant these cells into an irradiated mouse that lacks functional HSCs. ○ Observe if the labeled cells repopulate the blood and immune systems over time. Switch from Self-Renewing to Non-Self-Renewing: ○ Self-renewing: HSCs. ○ Non-self-renewing: Multipotent progenitors (MPPs), which lose self-renewal capability. 2. Effect of Making a BCR Protein Without the J Segment Without the J (Joining) segment: ○ The BCR would lack a functional antigen-binding site, as the J segment is critical for forming the variable region of the receptor. 3. Pathway of Rearrangement in B Cells Pro-B Phase: ○ Heavy chain rearrangement begins with VDJ recombination. ○ If successful, the cell proceeds to the next stage. Pre-B Phase: ○ Heavy chain expression is tested with a surrogate light chain. ○ Rearrangement of the light chain (VJ recombination) begins. Immature B Phase: ○ Both heavy and light chains are expressed on the surface as IgM. ○ The BCR is tested for self-reactivity. 4. Development Pathway of T Cells from ETP Pathway: ○ ETP (Early Thymic Progenitor) migrates to the thymus. ○ Double Negative (DN) Stage: Rearrangement of TCR beta chain begins. ○ Double Positive (DP) Stage: TCR alpha chain rearrangement occurs. Cells express both CD4 and CD8 markers. ○ Single Positive (SP) Stage: Cells differentiate into either CD4 (helper T cells) or CD8 (cytotoxic T cells). 5. Potential Errors During Recombination/Rearrangement  Errors: ○ Non-functional receptor due to improper V(D)J joining. ○ Self-reactive receptors that could cause autoimmunity. ○ Chromosomal translocations leading to cancer. 6. Determining Functional Receptor Protein Test functionality by expressing the receptor on the cell surface. Use antigen-binding assays to verify that the receptor binds to its target. A functional receptor will also trigger downstream signaling in response to antigen binding. 7. Outcome of Producing a Self-Reactive BCR Self-reactive BCRs undergo receptor editing (light chain rearrangement) or clonal deletion (apoptosis) to prevent autoimmunity. 8. Lineage Specificity Determinants: ○ Transcription factors (e.g., EBF1 for B cells, Notch1 for T cells). ○ Epigenetic modifications that silence genes of alternative lineages. Emergence of Different Cell Types: ○ B cells develop in the bone marrow, while T cells require signals in the thymus to commit to their lineage. 9. Differentiation Potential of Cell Populations HSC vs. MPP vs. B/T Progenitors: ○ HSCs: Fully multipotent and self-renewing. ○ MPPs: Multipotent but lack self-renewal. ○ B/T progenitors: Committed to a specific lineage, with reduced potential for differentiation. 10. Polymorphonuclear Cells Definition: ○ White blood cells (e.g., neutrophils) with multilobed nuclei. Development of Shape: ○ Nuclear segmentation occurs as these cells mature, aiding in their mobility and function in the immune response. 11. FACS Analysis: Spleen vs. Thymus Cells Definitions: ○ Forward Scatter (FSC): Measures cell size.  ○ Side Scatter (SSC): Measures cell complexity (granularity). Labeling Populations: ○ Spleen: Includes mature B and T cells (high FSC and SSC variability). ○ Thymus: Enriched with immature T cells (lower FSC and SSC). Additional Questions and Explanations 1. Immunoglobulin Gene Rearrangement Germline Configuration (Unrearranged): ○ In germline DNA, immunoglobulin genes are in their original state, with separated V (Variable), D (Diversity), and J (Joining) segments. Example: mathematica Copy code V1, V2, V3... Vn - D1, D2... Dn - J1, J2, Jn - Constant Region ○ Rearranged Configuration in Pre-B Cell: ○ After VDJ recombination, one V, one D, and one J segment are joined, forming a single contiguous sequence encoding the variable region. Example: mathematica Copy code V3 - D1 - J2 - Constant Region ○ ○ This rearrangement allows the formation of a functional heavy chain. 2. Differences Between ɑ/β and Ɣ/δ T Cells ɑ/β T Cells: ○ Most common T cell type (90–95% in peripheral blood). ○ Recognize antigens presented by MHC molecules (Class I or II). ○ Play central roles in adaptive immunity: CD4+ Helper T Cells: Coordinate immune responses. CD8+ Cytotoxic T Cells: Kill infected or cancerous cells. Ɣ/δ T Cells: ○ Less common, typically found in mucosal tissues.  ○ Recognize non-classical antigens without requiring MHC presentation. ○ Special Roles: Early responders to infection (innate-like function). Tissue surveillance and repair. 3. Experimental Design: Role of Transcription Factors in Lineage Specificity Objective: Determine if transcription factors (TFs) dictate lineage specificity during cell differentiation. Method: ○ Knockout or Overexpression Studies: Use CRISPR/Cas9 to knock out TFs known to influence lineage (e.g., EBF1 for B cells, Notch1 for T cells). Overexpress TFs in multipotent progenitors and observe changes in lineage commitment. ○ Reporter Assays: Insert fluorescent reporters linked to lineage-specific genes (e.g., B-cell genes like Ig heavy chain or T-cell genes like CD3). Track fluorescence changes in cells with and without specific TFs. ○ ChIP-Seq: Use chromatin immunoprecipitation followed by sequencing to identify DNA regions bound by TFs in differentiating cells. ○ Functional Testing: Transplant modified cells into irradiated mice to test their ability to differentiate and repopulate specific lineages. Outcome: ○ If TFs are removed or disrupted, loss of lineage-specific differentiation suggests they are essential for determining cell fate. Part 2 - Cancer: Key Questions and Answers 1. Experiment to Prove the Existence of Chromosome Territories Design: 1. Fix cells using paraformaldehyde to preserve chromatin structure. 2. Apply fluorescence in situ hybridization (FISH) with probes specific to individual chromosomes. 3. Observe using a confocal microscope to visualize spatially distinct regions occupied by each chromosome. Expected Outcome: Distinct fluorescence signals for each chromosome, demonstrating non-overlapping territories. 2. Driving Force of Chromatin Dynamics Key Forces: ○ Histone Modifications: Influence chromatin compaction (e.g., acetylation loosens, methylation tightens). ○ Nuclear Architecture: Nuclear lamina anchors heterochromatin; euchromatin occupies central regions. Separation of Euchromatin and Heterochromatin: ○ Euchromatin: Active, gene-rich regions positioned centrally for transcription. ○ Heterochromatin: Repressive regions associated with the nuclear periphery. 3. Enhancer Locations for Gene Expression Expressed Gene: ○ Enhancers loop close to the promoter region, facilitating transcription factor binding and transcription. ○ Located in euchromatin near the active transcription machinery. Non-Expressed Gene: ○ Enhancers are inaccessible within heterochromatin or spatially distant from the promoter. Location on the Rosette: ○ Enhancers for active genes are brought into transcriptional hubs, while inactive enhancers remain at the periphery of the loop structure. 4. Exceptions to the Exclusivity of Heterochromatin and Euchromatin  Examples: ○ Some highly expressed genes, like ribosomal RNA (rRNA) genes, are located near the nuclear lamina yet remain transcriptionally active. ○ Stress-response genes can dynamically shift between heterochromatin and euchromatin. 5. Function of Nuclear Pores Purpose: ○ Regulate transport of RNA, proteins, and other molecules between the nucleus and cytoplasm. ○ Facilitate transcriptional activity by tethering highly expressed genes near the pores. 6. Localization of Genes to Nuclear Pores Why Some Genes Localize: ○ Genes requiring rapid transcription and export (e.g., stress-response genes) are often tethered to nuclear pores. Why Others Do Not: ○ Repressed genes or those not requiring export remain in other nuclear regions. 7. Importance of Loops and Rosettes for Immunoglobulin Gene Rearrangement Role: ○ Loops bring V (Variable), D (Diversity), and J (Joining) segments into proximity to enable recombination. ○ Ensures efficient rearrangement necessary for antigen receptor diversity. Location: ○ V segments: Positioned distally. ○ D segments: Intermediate. ○ J segments: Located proximally to the constant region. 8. CD4/CD8 Stain FACS Plot in E47 Knockout (KO) Expected Plot: ○ Reduced populations of double-positive (CD4+CD8+) cells in the thymus, as E47 is crucial for T cell development.  ○ Single-positive CD4+ or CD8+ cells may also be reduced. 9. E47 in B and T Cell Development Explanation: ○ E47 activates lineage-specific target genes: In B cells, E47 promotes immunoglobulin gene rearrangement. In T cells, E47 regulates TCR gene rearrangement. ○ Lineage specificity is achieved through interactions with co-factors and chromatin accessibility unique to each lineage. 10. Experiment to Determine if Enlarged Thymus is Due to Inflammation or Cancer Method: 1. Perform histological analysis: Look for inflammatory markers (e.g., cytokines, immune cell infiltration) vs. abnormal cell proliferation. 2. Conduct flow cytometry: Analyze the cell populations (e.g., CD4/CD8 profiles). 3. Perform genomic analysis: Test for mutations or oncogene activation (e.g., Ras, p53). 11. Experiment to Prove Gene/Mutation Causes Tissue Transformation Steps: ○ Transfect primary cells with the gene/mutation of interest. ○ Observe for cancerous characteristics: Loss of contact inhibition. Ability to grow in soft agar (anchorage independence). ○ Compare with control cells lacking the mutation. ○ Outcome: If the mutation induces these traits, it is likely oncogenic. 12. Advantages of Cell Lines Over Primary Cells Cell Lines: ○ Easy to culture and maintain. ○ Divide indefinitely, providing a consistent model for experiments. Primary Cells: ○ Directly isolated from organisms but have limited growth potential. ○ Better represent in vivo conditions but are harder to manipulate and maintain.