Grnz B Delivery & Grnz B–Mediated Cell Death

Mechanisms of CTL Killing of Target cells (2)

Grnz B Delivery & Grnz B–Mediated Cell Death Apoptosome

What Happens Without CTL Killing Function?* Familial hemophagocytic lymphohistiocytosis (HLH): mutations in peforin gene, or other genes critical for degranulation of cytotoxic granules. Uncontrolled activation and proliferation of CD4+ and CD8+ T cells, cytokine storm, macrophage activation and proliferation, pancytopenia, and anemia. Activated macrophages in the spleen and bone marrow are intensely phagocytic, removing erythrocytes, leukocytes, and platelets from the circulation. Prf1−/− mice infected with LCMV or CMV develop a HLH-like syndrome * Or NK killing function

Clinical Evidence for Roles of CD8+ T cells CD8 T cell deficiency caused by homozygous mutations in CD8, TAP1, or TAP2 Chronic sinopulmonary infections  DOCK8 defciency (CD8 T cell and B cell defects) Severe cutaneous viral infections with HSV, human papillomavirus (HPV), and scarring Molluscum contagiosum  SAP deficiency -X-linked Lymphoproliferative syndrome (XLP): diminished NK and CD8+ T cells killing through 2B4, Persistence of EBV-infection, excessive NK and CD8+ T secretion of pro-inflammatory cytokines Exhausted CD8+ T cells in cancer patients Block PD-1, revive CTL, enhanced anti-tumor immunity and anti-viral responses 

Role of CTL/Perforin in Diseases Perforin plays a permissive role of in malaria: attack on antigen-bearing brain endothelial cells Perforin plays a key role in the autoimmune destruction of insulin-producing b cells in the pancreatic Islets leading to Type 1 diabetes mellitus CTL are major contributors to: Postviral myocarditis, and dilated cardiomyopathy Allograft rejection Hepatitis

CD8+ T cell Exhaustion

NK cells - What are they? 3rd lineage of “lymphocytes” Human CD3-,CD56+, (Nkp46+) Mouse CD3-,NKR-P1C+ (aka NK1.1), (NKp46+) 3rd lineage of “lymphocytes” Function in innate immunity to protect against viruses, bacteria, & tumors Produce cytokines & kill abnormal cells

NK Cells - Where do they come from? NK/T cell progenitor in bone marrow Thymus not required Do not rearrange TcR or Ig genes normal NK cells in scid and Rag-/- mice

NK Cells - Where do they live? ~5-20% peripheral blood lymphocytes ~5% lymphocytes in spleen, abundant in liver Low frequency in thymus, bone marrow, uninfected lymph nodes and lymphatics >70% of the lymphocytes in placenta

NK Cells - What do they do? Cell mediated-cytotoxicity – “natural killing” Antibody-dependent cellular cytotoxicity (ADCC) Early g-interferon production Secretion of other cytokines: TNFa, LTa, GM-CSF, IL-5, M-CSF, IL-3, IL-10, IL-13, MIP-1a, MIP-1b, RANTES, etc.

NK cells- How do they kill? Predominantly perforin + granzymes, just like CTLs Secreted or membrane TNFa Fas ligand TRAIL

NK cells Function Augmented by Cytokines **Interferon-a/b augments cytolytic activity **IL-15 required for development, induces proliferation, increases cytotoxicity **IL-12 & IL-18 augments INF-g production IL-2 induces proliferation, increases cytotoxicity…physiological relevance?

NK cells like to kill cells lacking MHC class I – “missing self” Karre et al. 1986 Nature 319:675

Activating and Inhibitory NK Cell Receptors (1) Fig. 4-6A

Activating and Inhibitory NK Cell Receptors (2)

Activating and Inhibitory NK Cell Receptors (3)

Inhibitory NK Cell Receptors

Activating NK Cell Receptors

NK Cells Kill Injured and Infected Cells

NK Cells Activate Macrophages

Innate Lymphoid Cells Cells with lymphocyte morphology that serve diverse antimicrobial functions. Arise from a common bone marrow precursor expressing ID2 transcription factor, and depend on IL-7 or IL-15 for development Unlike B and T cells, they emerge fully capable of performing effector functions without a need for clonal expansion and differentiation. ILCs use effector mechanisms shared by T cells, but do not express TCRs and do not rearrange antigen receptor genes. Several subsets of innate lymphoid cells, distinguished by their cell surface molecules, and by the effector mechanisms they use to perform their protective functions.

Innate Lymphoid Cells

Innate Lymphoid Cells Innate immunity (ILC) Adaptive immunity (CD4 T cells) Th1 cells (T-bet) Th2 cells (Gata3) Th17 cells (RORgt) TReg cells (Foxp3) Th22 cells (RORgt) IFN-g NK cells, ILC1 IL-13, IL-5 Nuocytes, ILC2 (RORa) IL-17 ILC17 cells (RORgt) IL-22 NK22 cells (RORgt) LTi cells (RORgt)

T cell activation states Naïve Activated Memory : central memory effector memory 10 2 3 4 5 < CD45RA> < CCR7 > 44 28.2 4.73 23.1 CM Naive EM TEM Naïve (CD45RA+CCR7+) CM: central memory (CD45RA-CCR7+) EM: effector memory (CD45RA-CCR7-) TEM: terminal effector memory (CD45RA+CCR7-)

2. TOLERANCE 1. Immune Mechanisms 3. Autoimmunity 4. Rheumatologic diseases Rheumatoid arthritis Systemic Lupus Erythematosus Spondarthropathies Inflammatory myopathies Systemic sclerosis Osteoarthritis

Tolerance Is……………. the immunologic unresponsiveness to self antigens It allows the immune system to protect the body without turning against itself The focus is on the adaptive immune system T & B cells must be able to discriminate self from non self This occurs centrally & peripherally

Central T Cell Tolerance T cells are produced in the bone marrow & migrate to the thymus. Here they go through a rigorous selections process. Only T cells that react to antigen but not self exit. The rest die by apoptosis. NEJM 2001;344(9): 655 – 664.

Peripheral T Cell Tolerance If autoreactive T cells enter the circulation, there are several mechanisms that can prevent an autoimmune reaction. NEJM 2001;344(9): 655 – 664.

B Cell Tolerance PERIPHERAL CENTRAL Clonal deletion of autoreactive B cells in the bone marrow, spleen & lymph nodes. PERIPHERAL Lack of help from T cells is the predominant factor.

Breakdown in peripheral tolerance

3. AUTOIMMUNITY Immune Mechanisms Tolerance 4. Rheumatologic diseases Rheumatoid arthritis Systemic Lupus Erythematosus Spondarthropathies Inflammatory myopathies Systemic sclerosis Osteoarthritis

Autoimmunity Breakdown in mechanisms preserving tolerance to self Severe enough to cause a pathological condition

Autoimmune diseases Organ specific e.g. Multisystem e.g. Insulin dependant diabetes Myasthenia gravis Multisystem e.g. Rheumatoid arthritis SLE

Mechanisms ENVIRONMENTAL FACTORS GENETIC FACTORS AUTOIMMUNE DISEASE Infectious/ noninfectious triggers Hypothesis : Molecular mimicry GENETIC FACTORS Aberant MHC/HLA - present self peptide Autoreactive T & B cells AUTOIMMUNE DISEASE Molecular mimicry : The antigen looks similar to a self-peptide. As a result, the body produces an immune response to the trigger factor as well as to self.

The Major Theories in the Development of Autoimmune Diseases Release of the normally sequestered antigens Increased expression of autoantigen/cryptic epitope/MHC II Molecular mimicry, Epitope spreading Defects in apoptosis Decreased cell numbers or function of suppressor and/or regulatory cells Altered Th1 and Th2 cytokine pattern Increased expression of costimulatory molecules Release of inflammatory mediators

Autoantibodies in Connective Tissue Diseases Produced by B cells May be pathogenic eg. Form immune complexes in lupus nephritis Markers of certain diseases Not diagnostic Apart from rheumatic disorders, they may be found in normal population & with other conditions Therefore only test when clinically indicated.

Autoantibodies associated with disease AUTOANTIBODY Rheumatoid Arthritis Rheumatoid factor SLE ANA,dsDNA, Smith Scleroderma ANA,centromere, topoisomerase Antiphospholipid Syndrome Anticardiolipin (ACLA) Sjogren’s syndrome Ro, La Polymyositis Jo-1 Dermatomyositis Mi-2 Wegener’s granulomatosis c-ANCA

Cellular Targets for autoantibodies Ab to intracellular proteins -proteinase 3 cANCA Ab to cell membrane Proteins ACLA Ab to IgG Rheumatoid factor Antinuclear antibodies (ANA) dsDNA ENA – Smith, Ro , La, RNP Centromere, topoisomerase Ribosomal & lysosomal components -t RNA synthetase AntiJo 1 This diagram depicts the autoantibodies & their respective target antigens

4. Rheumatologic conditions 1. Immune Mechanisms 2. Tolerance 3. Autoimmunity 4. Rheumatologic conditions Rheumatoid arthritis Systemic Lupus Erythematosis Ankylosing spondylitis Inflammatory myopathies (dermatomyositis, polymyositis) Systemic sclerosis (scleroderma) Osteoarthritis The above disease will be used to highlight some of the concepts of Immunology in Rheumatology. Note that the details of each pathway does NOT have to be memorized.

Rheumatoid Arthritis A symmetrical peripheral polyarthritis of unknown etiology that leads to joint deformity & destruction due to erosion of cartilage & bone

The immune mechanisms in RA Note: The interaction between the cells of the innate & adaptive immune systems The cytokines produced are targets for newer therapy in RA NEJM 2001; 344 (12): 907 – 916

RA The inflammatory process results in damage to cartilage & bone NEJM 2001; 344 (12): 907 – 916.

Rheumatoid Factor Rheumatoid Factor is an autoantibody produced in RA It is however produced in several other conditions  Clinical features are important in making the diagnosis

2003 Jun;3(3):323-8 Figure 1. Cartoon illustrating the major cell types and cytokine pathways thought to be involved in the destruction of bone and cartilage, mediated by IL-1 and TNFα, within the rheumatoid joint. The points at which the various antibody therapies discussed in the text are acting are marked by *. Ag, antigen; Th, T helper cell.

Systemic Lupus Erythematosis A generalized connective tissue disorder affecting many organs and characterized by the production of many autoantibodies

ACR Criteria for the diagnosis of SLE Note: 1.Many organs can be affected 2. Several auto- antibodies are associated with SLE

Systemic lupus erythematosus classification criteria (SOAP BRAIN MD) 1. Serositis: (a) pleuritis, or (b) pericarditis 2. Oral ulcers 3. Arthritis 4. Photosensitivity 10. Malar rash 11. Discoid rash 5. Blood/Hematologic disorder: (a) hemolytic anemia or (b) leukopenia of < 4.0 x 109 (c) lymphopenia of < 1.5 x 109 (d) thrombocytopenia < 100 X 109 6. Renal disorder: (a) proteinuria > 0.5 gm/24 h or 3+ dipstick or (b) cellular casts 7. Antinuclear antibody (positive ANA) 8. Immunologic disorders: (a) raised anti-native DNA antibody binding or (b) anti-Sm antibody or (c) positive anti-phospholipid antibody work-up 9. Neurological disorder: (a) seizures or (b) psychosis ". ..A person shall be said to have SLE if four or more of the 11 criteria are present, serially or simultaneously, during any interval of observation."

Lupus Nephritis The kidney biopsy on the right is from a patient with diffuse proliferative lupus nephritis shows massive deposits of IgG on immunofluorescence

Ankylosing Spondylitis (AS) AS is a chronic inflammatory disease of the axial skeleton (sacroiliac joint) manifested by back pain & progressive stiffness of the spine

Ankylosing Spondylitis The prevalence of the MHC,HLA-B27 is high in populations with Ankylosing Spondylitis

Dermatomyositis An idiopathic inflammatory myopathy associated with certain characteristic cutaneous manifestations Polymyositis – no cutaneous manifestation

Note: the inflammatory infiltrate in the muscle biopsy of this patient with Dermatomyositis

Scleroderma (systemic sclerosis) The term encompasses a heterogeneous group of conditions linked by the presence of thickened sclerotic skin lesions

The inflammatory process in Scleroderma results a marked fibrotic precess responsible for many of the clinical features

Scleroderma Lung Disease 2 important lung diseases which occur due to the inflammatory process in Scleroderma

References Sompayrac L. How the Immune System works. Blackwell Science, Inc. 1999 Roitt IM. Roitt’s Essential Immunology 10th ed. Blackwell Science 2001 Hochburg et al. Rheumatology 3rd ed. Mosby 2003 UpToDate 12.3 Kalla AA. Rheumatology Handbook. Rheumatic Diseases Unit Univrersity of Cape Town. 2003

References (cont) Parkin J, Cohen B. An overview of the immune system. Lancet 2001;357: 1777-1789. Mackay IR, Rosen FS. Tolerance and Autoimmunity. NEJM 2001;344(9): 655 – 664. Mackay IR, Rosen FS. Autoimmune diseases. NEJM 2001; 345(5): 340-350. Epstein FH. Cytokine pathway and Joint Inflammation in Rheumatoid Arthritis. NEJM 2001; 344 (12): 907 – 916. Yuan G et al. Immunologic Intervention in the Pathogenesis of Osteoarthritis. Arthritis & Rheumatism 2003; 48(3) 602- 611.