1. Grnz B Delivery & Grnz B–Mediated Cell Death

2. Mechanisms of CTL Killing of Target cells (2)

3. Grnz B Delivery & Grnz B–Mediated Cell Death
Apoptosome

4. 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

5. 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 

6. 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

7. CD8+ T cell Exhaustion

8. 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

9. 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

10. 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

11. 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.

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

13. 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?

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

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

16. Activating and Inhibitory NK Cell Receptors (2)

17. Activating and Inhibitory NK Cell Receptors (3)

18. Inhibitory NK Cell Receptors

19. Activating NK Cell Receptors

20. NK Cells Kill Injured and Infected Cells

21. NK Cells Activate Macrophages

22. 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.

23. Innate Lymphoid Cells

24. 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)

25. 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-)

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

27. 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

28. 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.

29. 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.

30. 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.

31. Breakdown in peripheral tolerance

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

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

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

35. 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.

36. 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

37. 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.

38. 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

39. 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

40. 4. Rheumatologic conditions
Immune Mechanisms
Tolerance
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.

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

42. 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

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

44. 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

45. 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.

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

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

48. 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 (c) lymphopenia of < 1.5 x (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."

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

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

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

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

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

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

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

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

57. 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

58. References (cont)
Parkin J, Cohen B. An overview of the immune system. Lancet 2001;357:
Mackay IR, Rosen FS. Tolerance and Autoimmunity. NEJM 2001;344(9): 655 – 664.
Mackay IR, Rosen FS. Autoimmune diseases. NEJM 2001; 345(5):
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)