Comprehensive University Study Notes on Medical Immunology and Immunotherapy

Fundamental Principles of the Immune System and Recognition Mechanisms

Immunology explores the biological defense mechanisms against invading pathogens, referred to as non-self, and the elimination of damaged self-components. The immune system is divided into two primary compartments: innate and adaptive. The innate system, also known as natural or native, acts as the first line of defense, intervening within minutes to hours ($0$ to $12$ hours). It constitutes physical, chemical, and microbiological barriers like the skin, the acidic $pH$ of the stomach, and the commensal microbiota which totals approximately $2\,kg$ in an adult. The innate response is aspecific, meaning its effector mechanisms are identical regardless of the specific bacterial or viral strain. It lacks immunological memory. Major cellular components include mononuclear phagocytes (macrophages), neutrophils, dendritic cells (APCs), mast cells, basophils, eosinophils, and Innate Lymphoid Cells (ILCs), such as Natural Killer (NK) cells. Soluble factors like the complement system, cytokines, and chemokines coordinate these actions.

The adaptive system, comprising T and B lymphocytes, provides a specific response with a slower kinetic ($7-10$ days for a primary response). These cells originate and mature in primary lymphoid organs—the bone marrow and thymus—and are activated in secondary lymphoid organs like the spleen, lymph nodes, and tonsils. Unlike innate cells, adaptive cells possess specific receptors, the TCR (T Cell Receptor) and BCR (B Cell Receptor), capable of recognizing distinct antigens. A vast receptor repertoire of approximately $10^9$ distinct specificities exists. Activated adaptive cells differentiate into effector cells to eliminate pathogens and memory cells to ensure rapid responses upon re-exposure.

Recognition in innate immunity relies on Pattern Recognition Receptors (PRRs) that bind to Pathogen-Associated Molecular Patterns (PAMPs) and Damage-Associated Molecular Patterns (DAMPs). PAMPs include molecular structures shared by many microorganisms, such as lipopolysaccharide (LPS) in Gram-negative bacteria, lipoteichoic acid in Gram-positive bacteria, and beta-glucans in fungi. DAMPs are endogenous molecules released by damaged or apoptotic cells. PRRs are germline-encoded and non-clonally distributed. They are located on cell membranes (e.g., certain Toll-Like Receptors or TLRs), endosomal membranes (detecting viral nucleic acids), or free in the cytoplasm (e.g., NOD-Like Receptors or NLRs, RIG-Like Receptors or RLRs, and cytoplasmic DNA sensors or CDS).

Signal Transduction Pathways and Intracellular Receptors

TLRs are glycoproteins characterized by extracellular leucine-rich repeats. TLRs $1, 2, 4, 5,$ and $6$ are typically found on the plasma membrane and recognize extracellular microbial components. TLRs $3, 7, 8,$ and $9$ are endosomal, recognizing viral RNA and bacterial DNA rich in unmethylated CpG islands. Binding triggers receptor dimerization and the recruitment of adapter proteins like MyD88 or TRIF. The MyD88-dependent pathway involves the activation of the transcription factor NF-kB, leading to the synthesis of pro-inflammatory cytokines such as TNF-$\alpha$, IL-1, and IL-6. The TRIF-dependent pathway, utilized by TLR3 and TLR4, activates Interferon Regulatory Factors (IRF3 and IRF7), inducing Type I Interferons ($\alpha$ and $\beta$) for antiviral responses. Type I Interferons are critical for SARS-CoV-2 defense, though uncontrolled responses can cause systemic tissue damage.

Cytosolic recognition pathways include the STING (Stimulator of IFN Genes) pathway. In response to cytosolic DNA (often from retrotranscribed viral RNA), the enzyme cGAS (Cyclic GMP-AMP Synthase) produces cGAMP, which binds to STING on the endoplasmic reticulum. This activates the kinase TBK1, phosphorylating IRF3 to induce Type I Interferon expression. RIG-Like Receptors (RLRs) specifically detect viral RNA, while NOD-Like Receptors (NLRs) comprise a family of approximately $20$ proteins. NLRs like NOD1 and NOD2 recognize bacterial peptidoglycan components like diaminopimelic acid (DAP) and muramyl dipeptide (MDP). Mutations in NOD genes are linked to inflammatory bowel diseases like Crohn's disease due to impaired production of bactericidal defensines.

The inflammasome is a multiproteic complex formed by NLR-P receptors (e.g., NLRP3), the adapter protein ASC, and pro-caspase-1. Activation leads to the cleavage of pro-caspase-1 into active caspase-1, which processes the precursors of pro-inflammatory cytokines IL-1$\beta$ and IL-18 into their active secreted forms. Hyperactivation of the inflammasome, triggered by factors like uric acid crystals or potassium efflux, underlies autoinflammatory syndromes such as Familial Mediterranean Fever and gout.

Inflammation and the Multi-Phase Process of Leukocyte Recruitment

Inflammation, or flogosi, is the temporary accumulation of immune cells and plasma proteins at the site of a stimulus. The recruitment of leukocytes from the blood to the inflamed tissue is a multi-step process. Cytokines like TNF and IL-1 activate vascular endothelial cells to express adhesion molecules. The first stage, rolling, is mediated by selectins (P-selectin or CD62P and E-selectin or CD62E). P-selectin is stored in Weibel-Palade bodies and expressed immediately, while E-selectin is synthesized after approximately $2$ hours. Leukocytes express ligands like PSGL1 and Sialyl-Lewis X (SLEX) that bind weakly to selectins, causing the cells to tumble along the vessel wall.

The second stage involves chemokines (e.g., CXCL8/IL-8) binding to G-protein-coupled receptors on the leukocyte surface. This triggers inside-out signaling, which induces a conformational change in leukocyte integrins (like LFA-1 and Mac-1), shifting them from a low-affinity to a high-affinity state. Simultaneously, cytokines increase the expression of integrin ligands like ICAM-1 (Intercellular Adhesion Molecule-1) and VCAM-1 (Vascular Cell Adhesion Molecule-1) on the endothelium. This results in firm adhesion. The final stage is diapedesis, or transendothelial migration, which can be paracelullar (between cells) or transcellular (through the endothelial cell). Alberto Mantovani proposed a "3-digit code" model for this process, where the first digit represents the selectin type, the second the chemotactic factor, and the third the integrin type. This specific combination determines which leukocyte subset extravasates.

Leukocyte Adhesion Deficits (LAD) are serious congenital immunodeficiencies. LAD-1 is caused by mutations in the $ITGB2$ gene ($CD18$ subunit for integrins). LAD-2 results from mutations in $SLC35C1$, leading to a fucose deficiency and defective Lewis acids for rolling. LAD-3 involves mutations in $FERMT3$ ($Kindlin-3$), affecting integrin activation and platelet aggregation. Clinically, these patients suffer from recurrent infections without pus formation (as neutrophils cannot reach the site) and marked peripheral neutrophilia.

Phagocytosis and Innate Immune Cell Specialization

Phagocytes include neutrophils, macrophages, and dendritic cells. Neutrophils are the most abundant leukocytes ($4-10 \times 10^6/mL$), with a short half-life of $2$ days. They contain primary (azurophilic), secondary, and tertiary granules containing proteases, metallo-proteases, and defensines. Macrophages originate from blood monocytes ($500,000-1 \times 10^6/mL$). Phagocytosis begins with the invagination of the plasma membrane to form a phagosome, which fuses with lysosomes to create a phagolysosome. The process is accompanied by an acidification of the internal $pH$ from $6.5$ to $4.5$, which activates lysosomal enzymes.

Elimination of pathogens is achieved through oxygen-dependent and oxygen-independent mechanisms. The oxygen-dependent mechanism involves the respiratory burst, mediated by the NADPH oxidase (phagocytic oxidase) complex. This complex consists of membrane subunits ($p22-phox$, $gp91-phox$) and cytosolic subunits ($p40-phox$, $p47-phox$, $p67-phox$). Activation results in the production of Reactive Oxygen Species (ROS) like superoxide anions and hydrogen peroxide ($H_2O_2$). Myeloperoxidase then produces hypohalites. Chronic Granulomatous Disease (CGD) involves mutations in these subunits (e.g., X-linked $gp91$ deficiency), preventing ROS production and leading to persistent bacterial infections and granuloma formation. Diagnosis uses the Nitroblue Tetrazolium (NBT) test or the Dihydrorhodamine (DHR) flow cytometry test. Pathogens like $Mycobacterium\,Tuberculosis$ can evade this by inhibiting phagosome-lysosome fusion or TACO protein removal.

Dendritic cells (DCs) are the bridge between innate and adaptive immunity. Langerhans cells in the skin act as sentinels. Upon capturing antigen, DCs downregulate PRRs and upregulate MHC II and co-stimulatory molecules (B7-1/CD80, B7-2/CD86). They migrate to lymph nodes via the CCR7 receptor in response to CCL19 and CCL21 chemokines. DCs find T cells in the parafollicular areas. Conventional DC1s (cDC1) are specialized in cross-presentation, allowing extracellular antigens (viral or tumor) to be loaded onto MHC I for CD8 T cell activation. Plasmacytoid DCs (pDC) are the major source of Type I Interferons.

The Major Histocompatibility Complex (MHC) and Antigen Presentation

The MHC (HLA in humans, chromosome $6$; H2 in mice, chromosome $17$) is the most polymorphic locus in the genome ($3500\,kb$ length). MHC Class I molecules (HLA-A, B, C) are expressed on all nucleated cells and consist of an alpha chain ($47\,kD$) and a non-covalently linked $\beta_2$-microglobulina ($12\,kD$, chromosome $15$). The peptide-binding groove (formed by $\alpha_1$ and $\alpha_2$ domains) is closed at both ends, accepting peptides of $8-11$ amino acids through specific "anchor residues." MHC Class II molecules (HLA-DP, DQ, DR) are expressed on professional APCs. They consist of an alpha and a beta chain, and their binding groove is open, accepting longer peptides ($15-30$ amino acids).

Antigen processing for MHC Class I usually involves cytosolic proteins. These are tagged with ubiquitin and degraded by the immunoproteasome (subunits LMP2, LMP7). Peptides are transported into the endoplasmic reticulum (ER) by TAP proteins. Tapasin helps load the peptide onto the MHC I molecule before it is shuttled to the Golgi and the cell surface. MHC Class II processing involves the endocytosis of extracellular proteins into vesicles where they are cleaved by cathepsins. In the ER, MHC II molecules are stabilized by the invariant chain, which blocks the binding site. A fragment called CLIP remains in the groove until HLA-DM exchanges it for the antigenic peptide in the endolysosome.

Heredity of HLA is codominant; offspring inherit one haplotype from each parent. Because of the vast polymorphism (over $31,000$ identified alleles), matching for transplants is difficult outside of families. Registries like IBMDR in Genoa facilitate marrow donations. Non-classical MHC I molecules include HLA-G (placental tolerance), HLA-E (NK cell regulation), and MICA/MICB (stress signals for NK and $\gamma\delta$ T cells). Certain HLA alleles are strongly associated with diseases, such as HLA-B27 with Ankylosing Spondylitis and HLA-DQ2/DQ8 with Celiac Disease.

Adaptive Immunity: T Lymphocyte Development and Polarization

T cells mature in the thymus, undergoing V(D)J recombination mediated by RAG-1 and RAG-2 proteins. Stem Cell Memory ($T_{SCM}$) and Central Memory ($T_{CM}$) cells reside in lymphoid tissues, while Effector Memory ($T_{EM}$) and Resident Memory ($T_{RM}$) cells migrate to peripheral tissues. $\gamma\delta$ T cells (specifically $V\gamma9V\delta2$) recognize non-peptide phosphoantigens like Isopentenyl Pyrophosphate (IPP) via the Butyrophilin (BTN3A1) molecule. Their activation is independent of MHC restriction. MAIT (Mucosal Associated Invariant T) cells recognize Vitamin B2 metabolites presented by the MR1 molecule. NKT cells recognize glycolipids presented by CD1d.

CD4 Helper T cell differentiation depends on the cytokine environment (the "third signal"). Th1 cells differentiate under IL-12 and IFN-$\gamma$ (activating T-bet/STAT4) to fight intracellular pathogens. Th2 cells require IL-4 (activating GATA3/STAT6) to counter parasites/helminths and are involved in allergies. Th17 cells differentiate via IL-6, IL-1, and IL-23 (activating ROR$\gamma$t/STAT3) to combat extracellular bacteria and fungi. T Follicular Helper (Tfh) cells express CXCR5 to enter follicles and help B cells. Regulatory T cells (Tregs), characterized by CD4, high CD25, and the FoxP3 transcription factor, maintain peripheral tolerance. They secrete inhibitory cytokines like IL-10 and TGF-$\beta$, and can consume available IL-2 with their high-affinity receptors to starve effector T cells of growth signals.

T cell activation requires three signals: 1) Antigen/MHC binding to TCR, 2) Co-stimulation (CD28 binding to B7-1/CD80 or B7-2/CD86), and 3) Cytokine polarization. Checkpoint molecules like CTLA-4 and PD-1 act as brakes. CTLA-4 competes with CD28 for B7 ligands with higher affinity, leading to anergy. S1P (Sphingosine-1-phosphate) gradients regulate T cell egress from lymph nodes; CD69 expression during activation transiently inhibits the S1P receptor ($S1PR1$) to keep the cell in the node for proliferation.

Adaptive Immunity: B Lymphocyte Activation and Immunoglobulins

B cells are classified as B1 (fetal liver derived, mucosal), Marginal Zone (non-circulating, splenic), and Follicular (circulating, protein-responsive). Repertoire specificity is generated by V(D)J recombination ($10^{15}$ possible BCRs). First-encounter antigens trigger a primary response characterized by low-affinity IgM. Secondary responses involve isotype switching and affinity maturation in germinal centers. Germinal centers have a dark zone where centroblasts undergo high proliferation and somatic hypermutation of the CDR regions, and a light zone where centrocytes interact with Tfh cells and Follicular Dendritic Cells (FDCs) to be selected for high affinity.

Immunoglobulins (Ig) consist of two light chains (kappa or lambda, $25\,kDa$) and two heavy chains ($50\,kDa$) arranged in a Y-shape. The Fab region binds antigen, while the Fc region mediates effector functions. Five classes exist:

• IgG: Most abundant ($13.5\,mg/mL$), crosses the placenta via the neonatal receptor ($FcRn$), mediates ADCC (Antibody-Dependent Cellular Cytotoxicity) via CD16.
• IgA: Secretory dimer, protects mucosal surfaces, contains a "secretory component" to resist proteases.
• IgM: Pentamer, first secreted in primary response, potent complement activator.
• IgD: Primarily a membrane receptor on naive B cells.
• IgE: Citophilic, binds high-affinity $Fc\epsilon RI$ on mast cells and basophils, triggers allergy mechanisms ($1\,ng/mL$ concentration).

The complement system is activated via three pathways: Classic (initiated by C1q binding to IgM or IgG), Lectin (MBL binding to mannose), and Alternative (spontaneous C3 hydrolysis). All pathways converge on the C3 convertase, leading to the formation of the Membrane Attack Complex (MAC, $C5b-C9$) which lyses the pathogen. Anaphylatoxins ($C3a$, $C4a$, $C5a$) increase vascular permeability and recruit leukocytes.

Immunological Tolerance, Hypersensitivity, and Autoimmunity

Central tolerance occurs in the thymus (T cells) and bone marrow (B cells). T cells that bind self-peptide with high affinity are deleted (negative selection). AIRE (Autoimmune Regulator) facilitates the expression of tissue-specific antigens in the thymus; mutations lead to APECED syndrome. B cells can undergo "receptor editing" to change specificity. Peripheral tolerance mechanisms include anergy (irreversible unresponsiveness due to lack of signal 2), clonal exhaustion (apoptosis via Fas/FasL), and suppression by Tregs. Sites like the brain and eyes are "immunologically privileged" through antigen sequestration.

Hyper-reactivity results in hypersensitivity. Type I is immediate/IgE-mediated (allergies, anafilassi). Diagnosis involves the Prick test or RAST (ELISA). Type II is cytotoxic (antibody/complement), seen in Hemolytic Disease of the Newborn ($Rh-$ mother, $Rh+$ fetus) or transfusion reactions. Type III involves immune complexes (e.g., SLE, glomerulonephritis). Type IV is delayed/cell-mediated ($48-72$ hours), seen in the Mantoux test for TBC and contact dermatitis. Type V is stimulatory (Baseow-Graves), Type VI is ADCC/Killer, and Type VII is inhibitory (Myasthenia gravis).

Autoimmunity involves a breakdown of tolerance. While some are monogenic (IPEX due to FoxP3 mutation, APECED due to AIRE), most are multifactorial. Molecular mimicry involves a foreign antigen resembling a self-protein, causing cross-reactivity. "Spreading" occurs when initial damage releases more self-antigens, widening the immune attack. TBC remains the leading infectious cause of death worldwide ($3\,million/year$); it creates a specific architecture called the tubercular granuloma to confine the latent bacterium. IGRA tests (ELISA/ELISPOT) measure IFN-$\gamma$ produce in response to specific ESAT-6 and CFP-10 proteins to detect latent infection without interference from the BCG vaccine.

Vaccinology and Advanced Cancer Immunotherapy

Vaccines aim to induce protective, long-lasting active immunity. History spans from Thucydides' observations to Jenner's smallpox vaccine and the current mRNA platforms. mRNA vaccines (e.g., for SARS-CoV-2) use lipid nanoparticles to deliver the Spike protein code; the RNA acts as its own adjuvant by triggering TLRs. Herd immunity (immunità di gregge) protects unvaccinated individuals in a population with high coverage. The "hygiene theory" suggests that reduced infectious disease exposure in the West has led to increased allergies and autoimmune issues.

Cancer immunotherapy leverages the immune system against tumors (Immunosurveillance). Tumors evade through MHC I downregulation, secreting TGF-$\beta$, or expressing PD-L1 to shut down CD8 T cells. Adoptive therapies include TIL (Tumor Infiltrating Lymphocytes) and CAR-T (Chimeric Antigen Receptor T cells). CAR-T cells are engineered to express a BCR-like receptor for a specific tumor marker (e.g., anti-CD19 for Leukemia). First-generation CARs used CD3$\zeta$; second-generation added co-stimulatory domains like CD28 or 4-1BB, drastically improving persistence but increasing the risk of Cytokine Release Syndrome (CRS).

Monoclonal antibodies are also used: Trastuzumab (anti-HER2) for breast cancer, Rituximab (anti-CD20) for lymphomas, and Ipilimumab (anti-CTLA4) as a checkpoint inhibitor. BiTEs (Bi-specific T-cell Engagers), like Blinatumomab ($19\,kDa$), bridge T cells and tumor cells. Although effective, BiTEs have a short half-life ($6\,hours$), requiring continuous infusion.

Questions & Discussion

Question: Does reducing the number of virus infections drastically have only positive effects, or can there be negative sides?

Response: An English immunologist developed the "hygiene theory," predicting that autoimmune and allergic pathologies are so prevalent in the Western world because infectious diseases are significantly reduced. The immune system evolved over thousands of years to provide protection against a wide range of agents. Th2 cells, specifically, guaranteed our survival against parasites for millennia. If parasites are gone, we cannot simply remove Th2 cells; thus, in the West, we have a pool of lymphocytes responsible for allergic pathologies. This is the price we pay. Ultimately, one must evaluate the cost-benefit ratio, noting that $13\,million$ people die annually from infectious diseases for which no vaccine or treatment currently exists.