Immune Cell Markers and Roles

Important Markers and Roles

• CD19 is present on all B cells (excluding plasma cells) and is part of the B cell coreceptor
• CD20 is present on all B cells except Pro-B and Pre-B cells
• CD40 is present on B cells and Dendritic Cells and interacts with CD40L to induce class switch recombination in B cells or trigger T cell activation in Dendritic cells
• MHC-I is present on all nucleated cells and presents peptide to CD8+ T cells
• MHC-II is present on APCs, especially dendritic cells, and presents peptide to CD4+ T cells
• Adhesion molecules are present on T cells (and others) to adhere to dendritic cells to maintain an immunological synapse
• TCR (alpha-beta or gamma-delta) is present on T cells
• Alpha-beta binds to MHC+antigen
• Gamma-delta binds to soluble antigen
• CD3 is present on all T-cells and is a transmembrane signal, coexpressed with TCR
• Zeta chains are present on all T-cells and are a transmembrane signal, coexpressed with TCR
• CD4 is present on Helper T cells and Regulatory T cells acts as a coreceptor, binding to the Beta-2 domain of class II MHCs
• CD8 is present on Cytotoxic T cells and acts as a coreceptor, binding to the Alpha-3 domain of class I MHCs
• CD14 is present on Monocytes/Macrophages but NOT dendritic cells
• B7 molecules (CD80 and CD86) are present on antigen-presenting cells and provide a second signal for T cell activation (if bound to CD28) or inhibition (if bound to CTLA-4)
• CD40L is present on Helper T cells and Interacts with CD40 to induce class switch recombination (B cells) or as part of the second signal to activate T cells
• CD28 is present on T cells and provides a second signal, binding to B7
• CTLA-4 is present on T cells and provides an inhibitory signal, binding to B7
• pre-T alpha receptor is present on Pre-T cells
• It is the Beta chain, but not the alpha chain, of the TCR with invariant chain (pre-Talpha)
• CD1d is present on APCs, functions like an MHC, and presents glycolipids to NKT cells instead of peptide
• CCR7 is present on T cells, B cells, Dendritic cells, and other cells as a chemokine, homing to secondary lymphoid tissues

Important Transcription Factors:

• NFAT (of NF-AT): T cell activation; IP3 triggers calcium release, which triggers calcineurin, which activates NFAT
• NF-kappaB, T cell activation: DAG activates Protein Kinase C, which triggers a pathway to activate it
• AP-1: T cell activation; DAG activates Ras which triggers MAP kinase cascade
• T-bet is associated with Th1 cells
• Gata 3 is associated with Th2 cells
• RoR gamma t is associated with Th17 cells
• FoxP3 is associated with Treg cells
• AIRE causes thymic cells to express and present non-thymic autoantigens

Cytokines:

• IL-2 is made by Activating T cells and promotes T-cell proliferation and Treg differentiation
• IL-12 is made by mature dendritic cells and switches to Th1, inducing T cell activation
• IFNy is made by Th1 cells, ILC1s, NKT cells, γδ, T cells, Tc, and switches to Th1, inhibits Th2 or Th17 switch, and activates killers, and isotype switch to IgG3
• IL-4 is made by mast cells, basophils, and Th2 cells, switching to Th2, inhibits Th1 or Th17, and induces isotype switch to IgE
• IL-5 and IL-13 are made by Th2 cells and ILC2s, causing allergy and asthma
• IL-6 is made by damaged Epithelial Cells and tissue-resident phagocytes (Module 2), which switches to Th7 and the inflammatory response from Module 2
• IL-17 and IL-22 is made by Th17 cells to activate inflammatory responses and wound healing
• TGF-β is made by Tregs, switching to Th17 (low) or Treg (high), suppressing immune responses
• IL-10 is made by Tregs, suppressing immune responses

Module 8: MHC and T Cells

• MHC class I presents on all nucleated cells, but not erythrocytes via a factory checkpoint
• All proteins present in the cell are presented, but T cells only respond to those that are not self-peptides
• MHC class I includes HLA-A, HLA-B, HLA-C
• MHC class II is primarily expressed on B cells, Macrophages, and Dendritic cells (Antigen Presenting Cells); other cells may express it when stimulated
• MHC class II Peptides are longer
• HLA includes HLA-DR, HLA-DQ, HLA-DP

MHC Restriction and Structure

• T cells cannot be activated without MHC-presenting antigen
• CD4 cells are Class 2 MHC-restricted
• CD8 cells are Class 1 MHC-restricted
• CD8 is restricted because T cells need either CD4 or CD8 coreceptors to activate CD8 is activated by binding to the α3 domain of Class I MHCs
• CD4 is restricted for the same reasons
• CD4 is activated by binding to the β3 domain of Class II MHCs
• Class 1 MHCs are expressed constitutively on all nucleated cells
• Class 2 MHC cells are expressed constitutively on APCs and thymic epithelial cells, and can be induced via cytokines on fibroblasts, endothelial cells, and T cells
• All class 1 MHCs will combine with β2-microglobulin (β2m)
• B2m acts as the 4th domain, but comes from a separate gene on a separate chromosome
• Antigen binds to the peptide-binding groove between a1 and a2.
• The Groove is “Cupped” and only fits 8-10 amino acids and typically has two anchors that determine what can bind
• 6 MHC genes is usually enough for all possible peptides

HLA Structure and MHC Pathways

• MHC-2 HLA Structure is composed of two separate genes that combine to make one protein
• Each polypeptide has two domains and cytoplasmic tails
• The #1 domain of each combines to make the peptide-binding groove
• Holds a longer peptide (13-25) and has ~3 anchors instead
• MHC Class 1 Pathway requires endogenous proteins only
• Only works via proteins MADE inside the cell
• As part of normal cellular processes, proteasome degrades all endogenous proteins into 15-amino acid peptides (15-mers)
• Peptides get further digested
• Some will move into the endoplasmic reticulum (ER) via peptide transporters TAP1 and TAP2
• If the right length, will be loaded into a newly synthesized MHC-I
• MHC with peptide goes to golgi and is processed for surface presentation
• Constant expression of multiple peptides in MHC-1
• CD8 T cells “scan” surface for their epitope
• MHC Class 2 Pathway includes exogenous proteins only, made outside the cell

Antigen Processing and Presentation

• Soluble antigen (toxins, lysed bacterial fragments, etc)
• Phagocytosed viruses or bacteria and vaccine components
• A and β chains are made separately and assembled with an invariant chain
• The invariant chain “caps” the groove so random peptides don’t bond yet
• The in variant chain is the guide– chaperones the MHC into the golgi apparatus, then into an acidic vessel which degrades MOST of the chain
• Small degraded CLIP remains until fusion
• HLA-DM swaps the CLIP for a new peptide.
• Process is selective– not every antigen will bind to every HLA

NK Cells

• Identifying
• CD3 Negative
• CD56 Positive
• Mature NK cells will express CD16
• Cell activation relies on a balance of activating and inhibiting signals
• NK cells get signals from Killer Ig-Like Receptors (KIR) and NKG2A
• KIR are highly polymorphic
• NKG2A recognizes nonstandard MHC Class I HLA-E which binds signal peptides from HLA-A, B, or C
• KIR cells recognize MHC-I directly (may be activating or inhibiting)
• KIRS+NKG2A inhibit NK cells
• Most healthy cells also lack activating triggers
• Stressed/Virally infected/Cancer cells display signs of distress on cell surface and/or secrete
• Natural Cytotoxicity Receptors (NCRs) bind to “stress” ligands to turn cells
• If anti-inflammatory signals regains strong, NK remains inactive
• If activation signals are strong and inhibition signals are weak, NK cell is activated
• Perforin/Granzyme release triggers apoptosis

HLA Nomenclature, TCRs and T Cell Development

• Alleles with an allele “group” are usually antigenically similar
• Specific protein may help for graft rejection
• Sometimes separators get omitted, especially in older documents
• For class 2 MHCs, there are two genes: HLA-DQ alleles all start with HLA-DQA1 or HLA-DQB1
• HLA-B*57:01-09 are all resistant to HIV
• HLA-DQB1*06 is associated with protection against COVID
• HLA-DQA1*05:01 is strongly associated with celiac disease
• Many things with B cells also apply to T cells like Antigen specificity, clonal selection, and Germ-line somatic cell rearrangement (VDJ)
• Big differences are the targets
• Helper T cells help and target OTHER CELLS
• Cytotoxic T cells eliminate intracellular pathogens
• α chain gene segments flank the δ gene segments
• If α is successful, δ is automatically excluded B cell vs T cell receptors - Similarities
• All chains have a V domain and at least one C domain
• TCR isotypes exist, but are functionally the same
• Same globular form with inter and intra disulfide bridges B cell vs T cell receptors - Differences
• TCR is smaller. 2 chains vs 4
• 1 antigen binding site vs 2
• No hinge region
• No Fab or Fc fragments
• No secreted form
• Antigen binding must interact with both MHC and peptide fragment
• CD4 and CD8 are coreceptors
• Same role as the B-cell coreceptor
• CD4 binds to MHC class II
• CD8 binds to MHC class I
• Coreceptors are NOT exclusive to T cells
• CD4 found on monocytes, macrophages, and DCs
• CD8 found on DCs and NK cells Coreceptor Function
• Enhance binding to host cells
• Restrict T cell activation to the correct MHC complex
  • CD4 to MHC-2, CD8 to MHC-1
  • Enhance signal transductioun (like the B cell coreceptor)
• NOT THE SECOND SIGNAL
• CD28 acts as a second signal
• Same role as CD40 on the B cell
• Binds to B7-1 or B7-2 (markers present on activated B-cells)
• CTLA-4 also binds to B7-1 and B7-2
• CD40L function/role
• Provides secondary signal for B cells
• Thymus-dependent activation
• Beta and delta have D segments
• Alpha and Gamma do not
• Early T-cells migrate to the Thymus prior to V(D)J rearrangement
• At this stage they are double-negative cells No CD4 or CD8; Equivalent to pro-B cells
• VDJ happens for γ, δ, and β genes
• Rag-1, Rag-2, and TdT are all expressed here but not elsewhere; α chain will recombine later
• γδ cells are done; They leave the thymus and proceed to the epithelial sites, with no central tolerance
• PreT cell Receptor lacks an α chain but is otherwise complete
• Use an invariant chain like B cells (here called pre-Tα), a critical checkpoint, stopping VDJ for beta chain, triggering activation of VJ for α genes
• Double Positive Cells (DP cells) have a Completed TCR (with alpha chain) triggering upregulated of both CD8 and CD4 T cell development order
• T cells need SOME autoimmunity. TCR must bind to self-MHCs because Alpha beta TCRs that DON’T a bind to MHC are bad
• Cells go through two rounds of training -Positive selection: pick cells that WILL react -Negative selection: pick cells that WON’T react too much

Positive selection

• DP cell binds to Thymic Epithelial Cell (TEC)
  • Binding to MHC-2 leads to CD4 phenotype
  • Binding to MHC-1 leads to CD8 phenotype
  • MHC-TCR binding is a necessary survival signal
  • Cells which fail to bind do not survive
• Leads to population of Single Positive (SP) Thymocytes that are restricted to one class of MHC; only ~10% of thymocytes pass
• Negative Selection, similar to B cell selection
• Thymocytes who interact with MHC with self antigens on thymic DCs or medullary TECs are driven to apoptosis, and survivors leave the thymus
• Alpha beta T cells are MHC-Restricted, CD4 or CD8, Self-tolerant

Natural Killer T Cells and Two Signal Method in T Cell Activation

“Weird” T cells: NKT cells

• Natural Killer T Cells are Small (~1%) subset of T cells with Nk cell markers
• They develop during selection when TCR responds to CD1d instead of MH, responding to glycolipids presented by CD1d
• This is a semi-innate response and the “Invariant” chain:i.e., only specific V and J segments used “Weird” T cells: Treg Cells
• About 10% of peripheral CD4+ cells Identified primarily CD25 and transcription factor FoxP3
• Most develop in thymus but are Autoreactive: Recognize Self + MHC and some are induced outside the thymus
• They Inhibit responses to self and foreign peptide, which is critical for tolerance

Module 10: The two-signal method

• TCR-MHC interaction is not enough
• Naive T cells must recognize peptide in MHC plus additional signals from costimulators with APCs
• Dendritic cells (The "professional" APC) provide most secondary stimulations
• Two types of Dendritic Cells
  • Myeloid Dendritic Cells (Major type)
  • Plasmacytoid Dendritic Cells secrete interferons (cytokines) in early immune responses

Identifying DC and T cell activation

• Dendritic Cells primarily isolated based off what they don’t express
• Do not express CD3, CD19, CD20 or CD56
• They are a HLA-DR subset that doesn’t express CD14
• Myeloi DCs are CD1c+

Immature mDCs

• Most dendritic cells are immature, located in tissues and express TLRs to sense for pathogens with a notable for low level of MHC class II expression
• They become mature after processing a pathogen, which Triggers upregulation of MHC-2, B7 family molecules, CCR7, and IL-12
• DC Homing; Mature DC now migrates to lymph nodes (CCR7), Travels to the closest "draining” lymph node. They Can now present peptide and provide secondary stimulation trigger as Naive T cells circulate through lymph nodes looking for mDC with their matching peptide (Takes ~ 8-10 hours)
• Activation of CD4+ T cells starts with The FIRST signal (the MHC-II and Peptide, + TCR and CD4) Affinity for MHC/TCR alone is weak: very slippery without CD4 to stabilize
• The second signal comes from co-activator pairs: B7 molecules (several forms) bind to CD28 (This increases CTLA-4 expression on T cells; CTLA-4 acts as an inhibitor of activation, CD40 binds to CD40L
• The LIGAND generally does the activating; The RECEPTOR is generally activated

Signal Process- It takes time!

• MHC-TCR binding enhances expression of CD40L, CD40L-CD40 upregulates CD40 leading to more CD40-CD40L binding
• CD40L-CD40 binding upregulates B7 with CD28 being constitutive (not affected; B7-1 or B7-2 binding to CD28 provides a second singal which ALSO upregulates CTLA-4
• Adhesion molecules keep T cells and APCs engaged while TCR “scans” for the MHC-2 with matching peptide

Process Steps

• Step 1 includes an Immature dendritic cell phagocytoses pathogen
• Step 2, the Dendritic cell becomes mature, upregulates MHC-2, B7s, and IL-12 while migrating through afferent lymph vessels to nearest lymph node
• Step 3 is when the Mature DC presents peptide leading CD4+ cells to adhere to DC for several hours and “scan” for MHC/Peptide complex; then On a match, MHC-2 and B7 bind to TCR (+CD4) and CD28; CD40 on DC also binds to CD40L on T cell to boost signal