Exam II Review - Drug Delivery Systems PDF

EXAM 2 REVIEW Carlos A. Barrero M.D. Assistant Professor School of Pharmacy-Temple University October 12th , 2023 Note: Please review notes in original slide decks. THE INNATE IMMUNE SYSTEM AND DRUG DELIVERY SYSTEMS Patrick Glassman, PhD September 19, 2024...

EXAM 2 REVIEW Carlos A. Barrero M.D. Assistant Professor School of Pharmacy-Temple University October 12th , 2023 Note: Please review notes in original slide decks. THE INNATE IMMUNE SYSTEM AND DRUG DELIVERY SYSTEMS Patrick Glassman, PhD September 19, 2024  WHAT ARE DRUG DELIVERY SYSTEMS?  Drug delivery systems (DDS) are a formulation strategy that are used to modulate the disposition of drugs by:  Prolonging pharmacokinetics  Minimizing uptake into off-target tissues  Selectively targeting to sites of disease  Most particle-based DDS are lipid or protein-based complexes ~80-200 nm in diameter  This is similar in size to a virus! Image from https://ainslielab.web.unc.edu CLINICALLY-USED DRUG DELIVERY SYSTEMS LIPOSOMES Protein Nanoparticle LIPID NANOPARTICLES  Nanoparticles are used clinically to deliver small molecule drugs, mRNA, and siRNA  Approved therapeutic areas include oncology, infectious diseases, COVID-19, and hereditary transthyretin-mediated amyloidosis  Most approved carriers are lipid-based  MOST DRUG DELIVERY SYSTEMS ARE LIPID- Doxil BASED  Liposomes and lipid nanoparticles that are clinically used are generally:  Spherical  50-100 nm in diameter  Composed of a mix of phospholipids and cholesterol  Coated with polyethylene glycol mRNA Vaccine  Viruses that infect humans are typically:  Spherical or filamentous  20-300 nm in diameter  Composed of a protein capsid that protects nucleic acids  Possibly coated with a lipid envelope XR Li, XH Cheng et al. J Ovarian Res. 15:96 (2022). S Daniel, Z Kis et al. Trends Biotechno. 40(10):1213-1228 (2022).  DDS CAN BE ENGINEERED TO EVADE OR HARNESS THE INNATE IMMUNE SYSTEM Key Points for Today: 1. Selection of appropriate DDS properties can bias particles towards or against immune recognition 2. This can be used to promote delivery to specific organs and/or cells and bias response to therapy F Zahednezhad, M Saadat, et al. J Control Rel. 305:194-209 (2019).  FIRST IMMUNOLOGICAL BARRIER: COMPLEMENT Key Points for Today: 1. The complement system functions very efficiently on surfaces (e.g, lipid membranes) 2. Release of C3a and C5a lead to further activation of the innate immune system and can cause anaphylactoid reactions 3. There are numerous regulators of the complement pathway that can prevent deposition of C3 and/or release of C3a/C5a including Factors H and I J Giang, MAJ Seelen, et al. Front Immunol. 9:639 (2018).  COMPLEMENT-ASSOCIATED DDS TOXICITIES  Complement deposits on DDS via several mechanisms (properidin, antibody, PRR)  Release of anaphylatoxins (C3a, C5a) can promote a significant response termed Complement Activation Related PseudoAllergy (CARPA)  Most severe symptoms are cardiovascular in nature, including systemic hypotension, pulmonary hypertension, edema, elevated thromboxane B2, etc.  Slow infusions appear to reduce the NM La-Beck, MR Islam, et al. Front Immunol. 11:603039 (2021). severity of CARPA  ENGINEERING DDS TO ELIMINATE CARPA  Attachment of a natural complement regulator (Factor I) to liposomes is a strategy to reduce complement-associated side effects  Factor I:  Reduces C3 deposition on liposomes  Reduces release of C3a and C5a in plasma  Eliminates liposome-induced cerebral hypoperfusion Z Wang, ED Hood, et al. Adv Mater. 34(8):e2107070 (2022).  MACROPHAGES ELIMINATE DDS FROM CIRCULATION  Macrophages in contact with the bloodstream are the primary elimination site for DDS  We refer to this as the reticuloendothelial system (RES)  Kupffer cells in the liver take a large fraction (>90%) of injected DDS  Spleen, lungs, and bone marrow also represent sites of elimination  Significant effort has been paid to evading macrophage-dependent W Ngo, S Ahed, et al. Adv Drug Deliv Rev. 185:114238 (2022). elimination  NEUTROPHIL MARGINATION  Neutrophils transiently associate with the vessel wall in organs including the lungs in a process termed margination  ~50% of the intravascular pool of neutrophils are in this marginated pool  Local inflammation and injury can lead to dramatic increases in this pool A Dahdah, J Johnson, et al. Front Cell Dev Biol. 10:795784 (2022). C Summers, SM Rankin et al. Trends Immunol. 31(8):318-324 (2010).  PATTERN RECOGNITION BY MARGINATED NEUTROPHILS  Under inflammatory conditions, marginated neutrophils are primed to respond to further danger  Following induction of inflammation in mice using lipopolysaccharide (TLR4 signal), the pulmonary marginated neutrophil pool responds to specific patterns  Nanomaterials displaying agglutinated protein on the surface have high levels of lung uptake  This process was complement-dependent JW Myerson, PN Patel, et al. Nat Nanotechnol. 17(1):86-97 (2022).  ISCHEMIC STROKE: NOT JUST LOSS OF BLOOD FLOW  The initial insult in ischemic stroke is vessel occlusion, typically by a blood clot  Primary treatment must focus on reperfusion (tPA, mechanical thrombectomy)  Secondary damage due to oxidative stress and inflammation can persist for weeks after stroke BCV Campbell, DA De Silva, et al. Nat Rev Dis Primers. 5, 70 (2019). Z Zhou, J Lu, et al. Pharmacol Ther. 191, 23-42 (2018).  THE BLOOD-BRAIN BARRIER IN STROKE  The blood-brain barrier exhibits time-dependent increases in permeability following ischemic stroke  Inflammatory processes in the acute and subacute phases include endothelial activation  This includes upregulation of cell adhesion molecules that S Bernardo-Castro, JA Sousa, et al. Front Neurol. 11, 594672 (2020). play a role in immune cell recruitment  CELLS INVOLVED IN POST-STROKE INFLAMMATION  Endothelial activation and chemokine release promotes migration of immune cells into the brain following stroke  These cells can promote tissue repair or exacerbate damage based on their phenotype  Shifting macrophages from M1 to M2 could promote tissue repair  Immune cells can be recruited from circulating pools or from lymphoid tissues AM Planas. Stroke. 49(9), 2261-2267 (2018).  TARGETING MIGRATING LEUKOCYTES TO TREAT INFLAMMATION **** 1.5 **** % Albumin Leakage 1.0 *** 0.5 0.0 Naive Sham TNF-  Brain inflammation is characterized by edema (fluid buildup), which can be tracked using albumin accumulation  Liposomes were loaded with a corticosteroid (dexamethasone) and targeted to leukocytes  Treatment effectively reversed edema and polarized macrophages to the anti-inflammatory M2 phenotype J Nong, PM Glassman, et al. ACS Nano. 17(14), 13121-13136 (2023). Granulocyte Macrophage-Colony Stimulating Factor and inflammation Kishore Pathivada PhD Student 09.23.2024  Cytokines Cytokines are glycoproteins, secreted by different immune cells in the body Important for cell signaling and communication Plays a vital role in initiating and controlling inflammation https://www.creative-diagnostics.com/cytokines-and-cytokine-receptors-elisa-kits.htm 18  Classification of cytokines Kupsa, T et al. (2012). The role of cytokines in acute myeloid leukemia: a systematic review. Biomedical Papers of the Medical Faculty of Palacky University in 19 Olomouc, 156(4).  G-CSF and M-CSF Granulocyte colony-stimulating factor (G-CSF or GCSF), is a glycoprotein secreted by many immune cells Functions as cytokine, hormone, and a type of colony-stimulating factor Stimulates bone marrow to produce granulocytes and stem cells A recombinant G-CSF, named Filgrastim is approved in 1991 for treatment of neutropenia It works by stimulating the body to increase neutrophil production Bachu RD et al. Oncology biosimilars: New developments and future directions. Cancer Rep (Hoboken). 2022 Nov;5(11):e1720 20  M-CSF Macrophage colony-stimulating factor (M-CSF), is a secreted cytokine & hematopoietic growth factor Involved in the proliferation, differentiation, and survival of monocytes, macrophages, and bone marrow progenitor cells GM-CSF Granulocyte–macrophage colony-stimulating factor (GM-CSF), a myelopoietic growth factor and pro- inflammatory cytokine During inflammation, polarizes mature myeloid cells into a pro-inflammatory phenotype (paracrine/autocrine function) expands and mobilizes progenitor myeloid cells to sites of inflammation (endocrine function) Increased percentages of GM-CSF-expressing leukocytes have been found in SARS-CoV-2 patients GM-CSF has been shown to be upregulated in autoimmune conditions (such as rheumatoid arthritis) Also, in severe acute respiratory syndrome (SARS), acute respiratory distress syndrome (ARDS), and cytokine release syndrome (CRS) Lang FM et al. GM-CSF-based treatments in COVID-19: reconciling opposing therapeutic approaches. Nat Rev Immunol. 2020 Aug;20(8):507-514. 21  Downstream signaling cascade of GM-CSF receptor Signaling downstream of the GM-CSF receptor in myeloid cells. Binding of GM-CSF to the alpha chain of the GM-CSF receptor (GMCSFR) leads to its dimerization with the signaling beta chain subunit. Beta chain-associated JAK2 then promotes receptor transphosphorylation and initiates downstream signaling. Depending on the sites of phosphorylation by protein kinases on the beta chain, specific adaptors are recruited to activate downstream signaling cascades such as the PI3K and MAPK pathways; recruitment of adapter protein 14-3-3 to phosphorylated Ser585 on beta chain leads to activation of the PI3K signaling while recruitment of Shc to phosphorylated Tyr577 leads to the activation of MAPK/ERK signaling. JAK2 bound to GM-CSFR can also directly activate STAT5 phosphorylation. Activation of PI3K downstream of GM-CSFR leads to myeloid cell survival whereas activation of MAPK/ERK and STAT5 induce proliferation of cells in addition to promoting their survival. 1. Kumar, A., Taghi Khani, A., Sanchez Ortiz, A., & Swaminathan, S. (2022). GM-CSF: A double-edged sword in cancer immunotherapy. Frontiers in immunology, 13, 901277. 22  GM-CSF and inflammation  Blue arrows mean ‘secretes’ Myeloid cells can be monocytes/macrophages or neutrophils  black arrows mean ‘acts on’  dotted arrows indicate ‘movement or differentiation’ Lang FM et al. GM-CSF-based treatments in COVID-19: reconciling opposing therapeutic approaches. Nat Rev Immunol. 2020 Aug;20(8):507-514. 23 Lang FM et al. GM-CSF-based treatments in COVID-19: reconciling opposing therapeutic approaches. Nat Rev Immunol. 2020 Aug;20(8):507-514. 24  I Patel J, et al. A randomized trial of anti-GM-CSF otilimab in severe COVID-19 pneumonia (OSCAR). Eur Respir J. 2023 Feb 2;61(2):2101870 25  Sequential Events for Lymphocyte Development 1 2 3 4 5  The process by which lymphocyte progenitors in the thymus and bone marrow differentiate into mature lymphocytes that populate peripheral lymphoid tissues is called lymphocyte development or lymphocyte maturation  The greatest proliferative expansion of lymphocyte precursors occurs after successfully rearranged the Ig heavy chain gene (B cell) or the TCR β chain gene (T cell). Sequential Events for Lymphocyte Development  Commitment of progenitor cells to the B lymphoid or T lymphoid lineage.  Proliferation of progenitors and immature committed cells at specific early stages of development, providing a large pool of cells that can generate useful lymphocytes.  The sequential and ordered rearrangement of antigen receptor genes and the expression of antigen receptor proteins. (The terms rearrangement and recombination are used interchangeably.)  Selection events that preserve cells that have produced functional antigen receptor proteins and eliminate potentially dangerous cells that strongly recognize self antigens.  Differentiation of B and T cells into functionally and phenotypically distinct subpopulations. B cells develop into follicular, marginal zone, and B-1 cells; and T cells develop into CD4+ and CD8+ αβ T lymphocytes, natural killer T (NKT) cells, MAIT cells, and γδ T cells. Multipotent stem cells give rise to distinct B and T lineages  The EBF, E2A, and Pax-5 transcription factors induce the expression of genes required for B cell development:  Rag-1 and Rag-2 proteins  Pre-B and the B cell receptor  Down stream signaling proteins  The Notch 1 and GATA3, signaling proteins induce the expression of genes required for T cell development: IgM Rag-1 and Rag-2 proteins IgD  IL-7 is required for the proliferation of T cell progenitors: Mutations in the IL-7 gene rise to an immunodeficiency disorder in called X-linked severe combined immunodeficiency disease (X-SCID)  Epigenetic mechanisms that regulate Lymphocyte Development  the methylation of DNA on certain cytosine residues that generally silences genes.  Posttranslational modifications of the histone tails of nucleosomes (e.g., acetylation, methylation, and ubiquitination) that may render genes either active or inactive depending on the histone modified and the nature of the modification.  Active remodeling of chromatin by protein machines called remodeling complexes that can also either enhance or suppress gene expression: * Commitment of developing T cells to the CD4 or CD8 lineage depends on epigenetic mechanisms that silence the expression of the CD4 gene in CD8+ T cells.  The silencing of gene expression by noncoding RNAs: Deletion of Dicer, a key enzyme in miRNA generation, in the T lineage results in a preferential loss of regulatory T cells (Treg) and the consequent development of an autoimmune phenotype  Selection Processes That Shape the B and T Lymphocyte Repertoires  The process of lymphocyte development contains numerous steps, called checkpoints.  Pre-antigen receptors and antigen receptors deliver signals to developing lymphocytes that are required for the survival of these cells and for their proliferation and continued maturation. The pre-antigen receptor is the first checkpoint during lymphocyte development.  In the next step of maturation, developing B and T cells express complete antigen receptors and the cells are selected for survival based on what these receptors recognize.  Cells that express useful antigen receptors may be preserved, and potentially harmful cells that strongly recognize self structures may be eliminated.  In the T cell lineage, positive selection ensures the maturation of T cells whose receptors recognize self major histocompatibility complex (MHC) molecules.  The expression of the coreceptor on a T cell (CD8 or CD4) is matched to the recognition of the appropriate type of MHC molecule (class I MHC or class II MHC, respectively).  Mature T cells whose precursors were positively selected by self MHC molecules in the thymus are able to recognize foreign peptide antigens displayed by the same self MHC molecules on antigen-presenting cells in peripheral tissues.  Checkpoints in Lymphocyte Maturation Apoptosis Apoptosis  Positive selection preserves receptor-expressing cells and is coupled to the generation of different B cell subsets  Negative selection is the process that eliminates developing lymphocytes whose antigen receptors bind strongly to self-antigens present in the generative lymphoid organs.  Epigenetic mechanisms that regulate Lymphocyte Development  the methylation of DNA on certain cytosine residues that generally silences genes.  Posttranslational modifications of the histone tails of nucleosomes (e.g., acetylation, methylation, and ubiquitination) that may render genes either active or inactive depending on the histone modified and the nature of the modification.  Active remodeling of chromatin by protein machines called remodeling complexes that can also either enhance or suppress gene expression: * Commitment of developing T cells to the CD4 or CD8 lineage depends on epigenetic mechanisms that silence the expression of the CD4 gene in CD8+ T cells.  The silencing of gene expression by noncoding RNAs: Deletion of Dicer, a key enzyme in miRNA generation, in the T lineage results in a preferential loss of regulatory T cells (Treg) and the consequent development of an autoimmune phenotype  Selection Processes That Shape the B and T Lymphocyte Repertoires  The process of lymphocyte development contains numerous steps, called checkpoints.  Pre-antigen receptors and antigen receptors deliver signals to developing lymphocytes that are required for the survival of these cells and for their proliferation and continued maturation. The pre-antigen receptor is the first checkpoint during lymphocyte development.  In the next step of maturation, developing B and T cells express complete antigen receptors and the cells are selected for survival based on what these receptors recognize.  Cells that express useful antigen receptors may be preserved, and potentially harmful cells that strongly recognize self structures may be eliminated.  In the T cell lineage, positive selection ensures the maturation of T cells whose receptors recognize self major histocompatibility complex (MHC) molecules.  The expression of the coreceptor on a T cell (CD8 or CD4) is matched to the recognition of the appropriate type of MHC molecule (class I MHC or class II MHC, respectively).  Mature T cells whose precursors were positively selected by self MHC molecules in the thymus are able to recognize foreign peptide antigens displayed by the same self MHC molecules on antigen-presenting cells in peripheral tissues. Stages of B Lymphocyte Maturation CD20 CD20 CD20 CD20 Stages of T Lymphocyte Maturation  The αβ T cells mature into CD4+ class II MHC–restricted or CD8+ class I MHC–restricted T cells Immunoglobulin Expression during B Lymphocyte Maturation  T Lymphocyte Maturation in the Thymus  Thymic stromal cells, secrete IL-7, a critical lymphopoietic Double + growth factor.  The movement of cells into and through the thymus is driven by chemokines. Double - Cortex: CCR9:CCL25 Medulla: CCR7:CCL19/21 The cell death (Apoptosis) is due to a combination of factors, including: Failure to productively rearrange the TCR β chain gene and thus to fail the pre-TCR/β, Failure to be positively selected by self MHC molecules in the thymus, Self antigen–induced negative selection. Adaptive defenses Humoral immunity Immunoglobulin Antigen-binding site H ea vy ch ai n Li gh tc ha in Hinge region Stem region Heavy chain Light chain variable region variable region Heavy chain Light chain constant region constant region Disulfide bond Pre-T Cell Receptor  the TCR β chain is expressed on the cell surface in association with an invariant protein called pre-Tα, along with CD3 and ζ proteins to form the pre-TCR complex.  TCR α gene expression in the double-positive stage leads to the formation of the complete αβ TCR.  Double-positive cells that successfully undergo selection processes go on to mature into CD4+ or CD8+ T cells Domains of Immunoglobulin Proteins variable domains constant domains CDRs : Complementary Determining regions Germline Organization of Human Immunoglobulin Loci Domains of the TCR Protein variable constant domains domains CDRs : Complementary Determining regions Germline Organization of Human TCR Human Immunoglobulin Repertoire Transcriptional Regulation of Immunoglobulin Genes Immunoglobulin Gene Recombination and Expression TCR Gene Recombination and Expression Lymphocytes Maturation Monoclonal Antibodies in Clinical Use Monoclonal Antibodies in Clinical Use International Nonproprietary Names (INNs) for Monoclonal Antibodies  INN system begun in 1950 by the World Health Organization (WHO) to provide a unique (generic) name to identify each pharmaceutical substance  INN system has important goals and benefits: Clear identification, safe prescription and dispensing of medicines to patients. Communication and exchange of information among health professionals and scientists worldwide. Revised monoclonal antibody (mAb) nomenclature scheme Geneva, 26 May 2017 Programme on International Nonproprietary Names (INN) © World Health Organization 2017  Limitations of monoclonal antibodies Not orally bioavailable: delivery by injection usually needed Optimal for extracellular targets; difficult intracellular delivery. Cannot penetrate the blood-brain barrier. Chemistry, Manufacturing and Controls (CMC) Issues: – Complex molecules produced by living cells; hard to characterize and to control batch-to-batch variation and stability Potential for immunogenicity: If recognized as foreign by the immune system, will trigger the formation of anti-drug antibodies (ADA). – A mouse antibody injected into a human will elicit a Human Anti-Mouse Antibody (HAMA) response; – Even human antibodies can trigger antibody responses in humans; HAHA responses. Anti-Drug Antibodies (ADA) in Patients  Recombinant Antibodies Re-engineered to reduce immunogenicity in humans Paul Carter Nat. Rev. Cancer, Nov, 2001. 118-129. Recombinant Antibodies: Anti-CD20  Rituximab was developed using cloning and recombinant DNA technology from human and murine (mice or rat) genes. It was originally FDA approved in 1997 for treatment of non-Hodgkin Lymphoma that was resistant to chemotherapy.  Ofatumumab is the first fully humanized monoclonal antibody that targets the CD20 molecule and is approved for the treatment of previously untreated patients with CLL.  Obinutuzumab is a glycoengineered antibody that demonstrated significantly higher efficacy over rituximab in B-cell malignancies such as CLL. The effect of the glycoengineering improves the binding of monoclonal antibodies with immune cells.  Rituximab mechanisms of action  Apoptosis  The antibody-dependent cell-mediated cytotoxicity (ADCC)  complement-mediated cytotoxicity (CMC ) Effector functions of antibodies  Second- and third-generation anti-CD20 mAbs  The second-generation anti- CD20 mAbs include ofatumumab, veltuzumab, and ocrelizumab. These are humanized to reduce immunogenicity.  The third-generation humanized CD-20 mAbs have an engineered Fc region to increase their binding affinity for the FcγRIIIa receptor. Three third-generation mAbs, AME-133v, PRO131921 and GA101, are undergoing active clinical development. Shundong Cang, et al., Journal of Hematology & Oncology 2012  Antigens Metabolites DNA RNA Protein ✹ Antigens are substances specifically bound by antibodies or T lymphocyte antigen receptors. Antigens that bind to antibodies include a wide variety of biologic molecules, including sugars, lipids, carbohydrates, proteins, and nucleic acids. This is in contrast to most T cell antigen receptors, which recognize only peptide antigens.  Antigenic Determinants ✹ Macromolecular antigens contain multiple epitopes, or determinants, each of which may be recognized by an antibody. Linear epitopes of protein antigens consist of a sequence of adjacent amino acids, and conformational determinants are formed by folding of a polypeptide chain.  Antigen-Antibody Complexes ✹ The relative concentrations of polyvalent antigens and antibodies may favor the formation of immune complexes that may deposit in tissues and cause damage. ✹ Antibody binding to antigen can be highly specific, distinguishing small differences in chemical structures, but cross-reactions may also occur.  T Lymphocytes Subset in the Thymus  Functional and phenotypic differentiation into CD4+CD8− or CD8+CD4− single-positive (SP) T cells occurs in the medulla of the thymus, and mature T cells are released into the circulation.  Some double-positive cells differentiate into CD4+CD8− regulatory T cells (Treg CD4+)  The selection of developing T cells is dependent on recognition of antigen (peptide–MHC complexes) in the thymus and is responsible for preserving useful cells and eliminating potentially harmful ones.  Positive selection is the process in which thymocytes whose TCRs bind with low avidity (i.e., weakly) to self peptide–self MHC complexes are stimulated to survive and to differentiate either into CD4+ T cells or CD8+ T cells Activation of naive and effector T cells by antigen Sequence of events in T cell responses Cytokines  CD4 Lymphocytes Functions CD8  Role of Costimulation in T Cell Activation The proliferation and differentiation of naive T cells require signals provided by molecules on APCs, called costimulators, in addition to antigen-induced signals  Costimulatory Pathways The interaction of CD40L on T cells with CD40 on APCs enhances T cell responses by activating the APCs. Mechanisms of T cell costimulation by CD28. Costimulation molecules of the CD28 family  Therapeutic Costimulatory Blockade  CTLA-4-Ig is an approved therapy for rheumatoid arthritis and transplant rejection.  Inhibitors of the CD40L:CD40 pathway are in clinical trials for transplant rejection and autoimmune diseases  Antibodies that block the CTLA-4 and PD-1 inhibitory receptors are approved for the immunotherapy of tumors. They work by preventing CTLA-4 or PD-1 from binding their ligands, reducing inhibition and enhancing T cell activation and enabling the cancer- bearing individual to mount more effective antitumor immune responses.  T cell recognition of a peptide – MHC complex The function of MHC molecules is to bind and display peptides for recognition by CD4+ and CD8+ T cells MHC recognition is also required for the maturation of T cells, ensuring that mature T cells are restricted to recognizing only MHC molecules with bound antigens MHC molecules are highly polymorphic, and variations in MHC molecules among individuals influence both peptide binding and T cell recognition. Antigen presenting cells (APC) capture antigens from its site of entry or production and bring it to the lymphoid organs where naïve T lymphocytes are located. Major histocompatibility complex (MHC) molecules are on cells identifying as self or non-self. There are two classes of MHC molecules MHC I found on all nucleated cells; MHC II found on macrophages, dendritic cells and B cells. Major histocompatibility complex (MHC)  Properties of Antigen Presenting Cells (APC) APC is the term used to refer to specialized cells that display antigens to lymphocytes. APCs express class II MHC molecules and other molecules involved in stimulating T cells and are capable of activating CD4+ T lymphocytes. Dendritic cells Most effective APCs for activating naïve T cells and therefore for initiating T cell responses. Macrophages and B lymphocytes; great for previously activated CD4+ helper T cells rather than for naïve T cells  Functions of APC Antigen is the first signal for the activation of naïve T cells, the additional stimuli that also activate naïve T cells are called second signals Co-stimulators are membrane bound molecules of APCs that function together with antigens to stimulate T cells Adjuvants are products of microbes, that enhance the expression of co- stimulators and cytokines and also stimulate the antigen-presenting functions of APCs. Properties and Functions of APCs  Common routes of antigens entry Role of Dendritic Cells Some antigens are transported in the lymph by APCs (primarily DCs) that capture the antigen and enter lymphatic vessels. Antigens that enter the bloodstream may be sampled by DCs that are in the spleen, or captured by circulating DCs and taken to the spleen. Resting tissue-resident DCs use receptors to capture, such as C-type lectins, that bind and endocytose microbes or microbial proteins and then process the ingested proteins into peptides capable of binding to MHC molecules. DCs can ingest antigens by pinocytosis, a process that does not involve specific recognition receptors but serves to internalize whatever molecules might be in the fluid phase in the vicinity of the DCs. The DCs are activated by cytokines, such as tumor necrosis factor (TNF), produced in response to the microbes. The activated DCs (also called mature DCs) lose their adhesiveness for epithelia or tissues and begin to express a chemokine receptor called CCR7 that is specific for two chemokines, CCL19 and CCL21, that are produced in lymphatic vessels and in the T cell zones of lymph nodes.  Role of dendritic cells in antigen capture and presentation DCs are strategically located at the common sites of entry of microbes and foreign antigens (in epithelia) and in tissues that may be colonized by microbes. DCs express receptors that enable them to capture and respond to microbes. DCs migrate from epithelia and tissues via lymphatics, preferentially into the T cell zones of lymph nodes, and naive T lymphocytes also circulate through the same regions of the lymph nodes. Mature DCs express high levels of peptide-MHC complexes, co-stimulators, and cytokines, all of which are needed to activate naive T lymphocytes.  Map Of The Human MHC Genes  In humans, the MHC is located on the short arm of chromosome 6 and occupies a large segment of DNA, extending about 3500 kilobases (kb)  There are three class I MHC genes called HLA-A, HLA-B, and HLA-C, which encode three types of class I MHC molecules with the same names.  There are three class II HLA gene loci called HLA-DP, HLA-DQ, and HLA-DR. Each class II MHC molecule is composed of a heterodimer of α and β polypeptides.  The set of MHC alleles present on each chromosome is called an MHC haplotype.  MHC molecule expression Class I molecules are expressed on all nucleated cells They provide a display system for viral and tumor antigens, so these antigens can be recognized by CTLs and the antigen-producing cells can be killed Class I molecule expression is increased by the type I interferons IFN- and IFN- , which are produced during the early and innate immune response to many viruses Class II molecules are expressed only on dendritic cells, B lymphocytes, macrophages, thymic epithelial cells ad a few other cell types Class II molecules are regulated by cytokines and other signals in different cells. IFN - is the principal cytokine involved in the stimulating expression of class II molecules in APCs such as DCs and macrophages. IFN- provides a mechanism by which innate immunity promotes adaptive immunity, by increasing class II MHC expression on APCs, and provides an amplicafication mechanism in adaptive immunity.  Enhancement of class II MHC molecule expression by interferon-γ  IFN-γ, produced by NK cells and other cell types during innate immune reactions to microbes or by T cells during adaptive immune reactions, stimulates class II MHC expression on APCs and thus enhances the activation of CD4+ T cells.  IFN-γ and type I interferons have a similar effect on the expression of class I MHC molecules and the activation of CD8+ T cells.  Structure of MHC Molecules Each MHC molecule consists of an extracellular peptide-binding cleft, followed by an immunoglobulin (Ig)–like domain and transmembrane and cytoplasmic domains. The polymorphic amino acid residues of MHC molecules are located in and adjacent to the peptide-binding cleft. The nonpolymorphic Ig-like domains of class II and class I MHC molecules contain binding sites for the T cell molecules CD4 and CD8, respectively  Class I MHC Molecules  Class I molecules are composed of a polymorphic α chain noncovalently attached to the nonpolymorphic β2- microglobulin (β2m).  The ribbon diagram (right) shows the structure of the extracellular portion of the HLA-B27 molecule with a bound peptide.  Class II MHC Molecules  Class II molecules are composed of a polymorphic α chain noncovalently attached to a polymorphic β chain.  The ribbon diagram (right) shows the structure of the extracellular portion of the HLA-DR1 molecule with a bound peptide.  Polymorphic residues of MHC molecules The polymorphic residues of class I molecules are confined to the α1 and α2 domains, where they contribute to variations among different class I alleles in peptide binding and T cell recognition. The polymorphic residues of class II molecules are located in the α1 and β1 segments, in and around the peptide- binding cleft, as in class I MHC molecules  The mechanisms of antigen processing are designed to generate Processing of Protein Antigens peptides that have the structural characteristics required for associating with MHC molecules, and to place these peptides in the same cellular location as newly synthesized MHC proteins with available peptide-binding clefts.  Proteins that are present in the cytosol are degraded by proteasomes to yield peptides that are displayed on class I MHC molecules, while proteins that are ingested from the extracellular environment and sequestered in vesicles are degraded in lysosomes (or late endosomes) to generate peptides that are presented on class II MHC molecules The Class I MHC Pathway for Processing and Presentation of Cytosolic Proteins The Class II MHC Pathway for Presentation of Proteins Degraded in Lysosomes  Most class II MHC–associated peptides are derived from protein antigens that are digested in endosomes and lysosomes in APCs  Internalized proteins are degraded enzymatically in late endosomes and lysosomes to generate peptides that are able to bind to the peptide- binding clefts of class II MHC molecules.  Cross Presentation  Cells infected with intracellular microbes, such as viruses, are ingested by dendritic cells, and the antigens of the infectious microbes are transported into the cytosol and processed in proteasomes and presented in association with class I MHC molecules to CD8+ T cells. Thus, dendritic cells are able to present endocytosed vesicular antigens by the class I pathway. Note that the same cross-presenting APCs may display class II MHC–associated antigens from the microbe for recognition by CD4+ helper T cells Nature of T Cell Responses  Cytosolic antigens are presented by nucleated cells to CD8+ CTLs, which kill (lyse) the antigen-expressing cells.  Extracellular antigens are presented by macrophages or B lymphocytes to CD4+ helper T lymphocytes, which activate the macrophages or B cells and eliminate the extracellular antigens.  Immunodominance Of Peptides  Protein antigens are processed to generate multiple peptides; immunodominant peptides are the ones that bind best to the available class I and class II MHC molecules. The illustration shows an extracellular antigen generating a class II–binding peptide, but this also applies to peptides of cytosolic antigens that are presented by class I MHC molecules  Immune Receptor Family Immune receptors that activate immune cells have separate polypeptide chains for recognition and associated polypeptide chains that contain cytosolic Immunoreceptor tyrosine-based activation motifs (ITAMs). B cell receptor (BCR), the T cell receptor (TCR), and the high- affinity receptor for IgE (FcεRI) have ITAMs motifs. Inhibitory receptors in the immune system typically have ITIM motifs on the cytosolic portion of the same chain that uses its extracellular domain for ligand recognition. FcγRIIB is an inhibitory receptor found on B cells and myeloid cells. PD-1, an inhibitory receptor found on T cells, also has an immunotyrosine based “switch” motif in its cytoplasmic tail  T Cell Receptor for Antigen  The antigen receptor of MHC- restricted CD4+ helper T cells and CD8+ cytotoxic T lymphocytes (CTLs) is a heterodimer consisting of two transmembrane polypeptide chains, designated TCR α and β, covalently linked to each other by a disulfide bridge between extracellular cysteine residues  The antigen-binding portion of the TCR is formed by the Vβ and Vα domains.  The hypervariable segment loops that form the peptide- MHC binding site are at the top  Binding of TCR to a MHC molecule  The V regions of the TCR α and β chains contain short stretches of amino acids where the variability between different TCRs is concentrated, and these form the hyper- variable or complementarity- determining regions (CDRs)  Three CDRs in the α chain and three similar regions in the β chain together form the part of the TCR that specifically recognizes peptide-MHC complexes. Components of the TCR Complex  TCR Component CD4 and CD8 are T cell coreceptors that bind to nonpolymorphic regions of MHC molecules and facilitate signaling by the TCR complex during T cell activation. Mature αβ T cells express either CD4 or CD8 but not both. CD8 and CD4 interact with class I and class II MHC molecules, respectively, and are responsible for the class I or class II MHC restriction of these classes of T cells The Immune Synapse  The Immune Synapse The synapse forms a stable contact between an antigen-specific T cell and an APC displaying that antigen and becomes the site for assembly of the T Cell signaling machinery of the T cell, including the TCR complex, coreceptors, costimulatory receptors, and adaptors. The immune synapse provides a unique interface for TCR triggering, thus facilitating prolonged and effective T cell signaling. The synapse ensures the specific delivery of secretory granule contents and cytokines from a T cell to APCs or to targets that are in contact with the T cell. APC The synapse, may also be an important site for the turnover of signaling molecules. This degradation of signaling proteins contributes to the termination of T cell activation.  Signal Transduction Phosphorylation of proteins and lipids plays a central role in the transduction of signals from the TCR complex and coreceptors. Even before TCR activation, there is some basal tyrosine phosphorylation of ITAM tyrosines and some recruitment of ZAP-70, to these phosphorylated ITAMs.  Within seconds of TCR ligation, Lck phosphorylates Calcium- and Protein Kinase C-Mediated Signaling Pathways in T Lymphocytes  Activation of Transcription Factors That Regulate T Cell Gene Expression Different transcription factors are activated by different cytoplasmic signal transduction pathways, and the requirement for multiple transcription factors accounts for the need to activate many signaling pathways after antigen recognition; including those encoding cytokine receptors and effector molecules. Three transcription factors that are activated in T cells by antigen recognition and appear to be critical for most T cell responses are nuclear factor of activated T cells (NFAT), Changes in surface molecules after T cell activation T cell-mediated immune responses  Role of T cells in eradicating infections CD4+ T cells recognize antigens of phagocytosed and extracellular microbes and produce cytokines that recruit and activate the phagocytes to kill the microbes. CD8+ T cells can also secrete some cytokines and participate in similar reactions. B, CD8+ cytotoxic T lymphocytes (CTLs) recognize antigens of microbes residing in the cytoplasm of infected cells and kill the cells. Subsets of CD4+ Effector T Cells CXCR3 / CCR5 P, E Selectins Tissues of innate Immunity CCR3-4-8 Mucosal Tissues CCR6 Tissues cells, Macrophages Th1/Th2 Germinal centers Structure of cytokine receptors  JAK-STAT signaling induced by cytokines  Cytokine receptors of the type I and type II receptor families engage signal transduction pathways that involve non-receptor tyrosine kinases called Janus kinases (JAKs) and transcription factors called signal transducers and activators of transcription (STATs).  Small molecule JAK antagonists have been approved for the treatment of acute myeloid leukemia, and chronic inflammatory diseases, including rheumatoid arthritis and psoriasis.  T cell proliferation, triggered by cytokines such as IL-2, is targeted by some immunosuppressive small molecules. Rapamycin (mTOR inhibitor)  Biologic actions of IL-2  Autocrine  Paracrine  Endocrine Signaling through the TNF receptor Development of Subsets of CD4+ Effector T Cells Th1 Cells Th2 Cells Th17 Cells Functions of Th1 cells  IFN-γ activates macrophages to kill phagocytosed microbes.  FN-γ promotes the differentiation of CD4+ T cells to the Th1 subset and inhibits the development of Th2 and Th17 cells.  IFN-γ stimulates expression of several different proteins that contribute to enhanced antigen presentation and T cell activation  IFN-γ acts on B cells to promote switching to certain IgG subclasses, and to inhibit switching to IL- 4–dependent isotypes, such as IgE.  Th1 cells produce tumor necrosis factor (TNF) and various chemokines, which contribute to the recruitment of leukocytes and enhanced inflammation.  Th1 cells are also produce IL-10, which functions mainly to inhibit dendritic cells and macrophages and thus to suppress Th1 activation. Macrophage activation by Th1 cells Functions of Th2 Cells Classical and alternative macrophage activation Functions of Th17 Cells Role of helper T cells in the differentiation of CD8+ T lymphocytes IL-2 Paracrine Inhibition of CD8+ T Cell Responses: T Cell Exhaustion Steps in CTL-mediated lysis of target cells Mechanisms of CTL-mediated killing of target cells  Cancer Immunotherapy Interferon Interferon is also called interferon alfa or Intron A. Interferon is used for several different types of cancer including kidney cancer (renal cell cancer), Cancer melanoma, multiple myeloma, some types of Immunotherapy leukemia Interleukin 2 Aldesleukin is also called Interleukin 2, IL2 or Proleukin. IL2 is used to treat kidney cancer, currently in clinical trials for other cancer. Administration of interferon and IL-2 is subcutaneously or infusion, 3 times a week. The side effects of interferon and IL-2 include: a drop in blood cells causing an increased risk of infection, bleeding problems, tiredness and breathlessness, flu-like symptoms, diarrhea, tiredness and weakness (fatigue), feeling sick, loss of appetite, low blood pressure. https://www.cancerresearchuk.org/immunotherapy/types/cytokines