1. Cellular Basis of the Immune Response
Robert Fleischmann, Ph.D.
Professor
Department of Urologic Surgery
Office: G144 Diehl Hall
Telephone Number: (612)
Address:

2. Objectives To understand the major players in adaptive immunity
To understand the nature and general functions of cytokines
To understand the identity and functions of the cytokines that are most important for adaptive immunity
To understand the differential regulation for induction of Th1 and Th2 cells
To understand the general features of cytokine/cytokine receptor interactions
To understand the mechanisms by which interferons, as model cytokines, activate their target cells
To understand the positives and negatives of the clinical use of interferons, as model cytokines

3. Cells of the Immune System

4. Overview of Major Players of Adaptive Immunity
Dendritic cells, macrophages, and B cells are professional antigen-presenting cells.
CD4+ T cells (Th cells) are exposed to antigen bound to MHC Class II on antigen-presenting cells and are driven to differentiate into Th1 or Th2 cells.
Th1 cells drive the differentiation and proliferation of CD8+ cytotoxic T cells (CTLs).
Th2 cells drive the differentiation and proliferation of B cells to become antibody-producing plasma cells.

5. Activated Lymphocytes Differentiate Into Effector Cells
Note how the lymphocyte enlarges as it differentiates.
Note that T and B lymphocytes cannot be visually distinguished from each other until they are fully differentiated to their respective effector cells.

6. Lymphocyte Differentiation and Proliferation Is Driven by Cytokines
Cytokines are proteins that help the cells of the immune system to communicate with one another.
Cytokines are usually soluble mediator molecules, though some may remain cell-bound.

7. Cytokines Are Often Named for the Type of Cell that Produces Them
Macrophages: monokines
Lymphocytes: lymphokines or interleukins
Generic: cytokines

8. Individual Cytokines Activate Target Cells by Binding to Their Specific Receptors
Each cytokine binds to its unique receptor.
Binding affinities are very strong, with dissociation constants in the range of to (Abs are in the range of 10-7 to 10-10).
The receptor may be composed of multiple subunits with one specific subunit and one or more common subunits (also shared as subunits of receptors for other cytokines).

9. Cytokine Receptor Expression May Be Regulated
Some cytokine receptors are constitutively present but may be down-regulated by their specific cytokine ligand or by other cytokines (example: IFN receptors).
Some cytokine receptors are induced by treatment of target cells with other cytokines (example: IL-1 induces resting T cells that express the intermediate-affinity IL-2 receptor [composed of and  chains] to express the high-affinity receptor [composed of , , and  chains]).

10. Cytokine Cell Targets
Autocrine action: the target cell of the cytokine is the same as the producing cell.
Paracrine action: the target cell of the cytokine a nearby cell that is different from the producing cell.
Endocrine action: the target cell of the cytokine is a distant cell that is reached through the circulation.

11. Autocrine Action of Cytokines
1. A Th1 cell is stimulated to
produce mRNA for IL-2.
2. The IL-2 mRNA is transported
to the cytoplasm where it is
translated into IL-2 protein.
3. The IL-2 is secreted.
4. The IL-2 binds to a receptor
on the producing Th1 cell,
activating it.
CD4+ Th1 cell

12. Paracrine Action of Cytokines
CD8+ T cell
1. A Th1 cell is stimulated to
produce mRNA for IFN-.
2. The IFN- mRNA is transported
to the cytoplasm where it is
translated into IFN- protein.
3. The IFN- is secreted.
4. The IFN- binds to a receptor
on the CD8+ T cell,
activating it.
CD4+ Th1 cell

13. Endocrine Action of Cytokines
1. A macrophage is stimulated to
produce mRNA for GM-CSF.
2. The GM-CSF mRNA is
transported to the cytoplasm
where it is translated into GM-
CSF protein.
Stem Cell
3. The GM-CSF is secreted.
4. The GM-CSF travels through
the blood to the bone marrow
where it binds to a receptor on
a hematopoietic stem cell,
activating it.
Macrophage

14. General Properties of Cytokines
Cytokines secretion is generally a brief, self-limiting event.
Secreted cytokines generally have their greatest effects in the immediate area where they are produced.
Cytokines are active at picomolar concentrations.

15. Cytokine Secretion Is a Brief, Self-Limiting Event
Induction of cytokine mRNAs is a transient event.
In general, cytokine mRNAs are unstable and have a short half-life.
After their translation, most cytokines are rapidly glycosylated and secreted.

16. Individual Cytokines May Be Produced by A Single Cell Type or by Many Cells Types
IL-2 is produced by Th1 cells.
IL-4 is produced by Th2 cells.
Multiple Cell Types
GM-CSF can be produced by T cells, macrophages, endothelial cells, and a wide range of fibroblasts.
IFN- can be produced by virtually all fibroblasts and epithelial cells.

17. Individual Cytokines May Act on a Single Cell Type or on Many Cell Types
IL-10 acts on macrophages to block IL-12, subsequently blocking Th1 cells.
IL-12 acts on Th1 cells.
Multiple Cell Types (Pleiotropism)
IFNs act on multiple cell types.
IL-1 acts on T cells, B cells, macrophages, endothelial cells, fibroblasts, and epithelial cells.

18. An Individual Cytokine May Have Multiple Effects on a Target Cell: Pleiotrophy
IFN-
Induces antiviral proteins
Induces antiproliferative effects
Induces MHC Class I antigens
Stimulates NK cell activation
Stimulates IL-12 production.

19. Different Cytokines Can Share Similar Activities (Redundancy)
IFN-, IFN-, and IFN- share similar activities, while retaining some unique properties.
IL-1, TNF, and IL-6 can all cause fever.
IL-2 and IL-15 share similar abilities to activate T cell proliferation and differentiation, while retaining some unique properties.

20. Individual Cytokines May Influence the Action of Other Cytokines
They may have antagonistic effects.
IL-12 stimulates Th1 cell activation and proliferation, while IL-10 blocks macrophage production of IL-12 and therefore blocks Th1 cell activation and proliferation
They may have additive effects.
IFN- plus IFN-have additive effects.
They may have synergistic effects.
IFN- plus IFN- or IFN- plus IFN- have synergistic effects.

21. Cytokine Attributes

22. Cytokines Belong to Five Families
IL-1 family
Hematopoietin family
Interferon family
Tumor necrosis family
Chemokine family

23. Most Cellular Responses to Cytokines Require mRNA and Protein Synthesis
Cytokine binds to receptor.
A transmembrane alteration occurs allowing the intracellular part of the receptor to be activated.
Second messenger molecules are activated.
Second messenger molecules directly or indirectly activate transcription of specific genes.
Translation of the mRNA for the specific genes results in new cell function.

24. Many Cytokines Act as Growth Regulators
CSFs stimulate bone marrow stem cells to grow.
IL-2 and IL-15 stimulate Th1 cells to grow and IL-2 stimulates Th2 cells to grow.
IL-10 blocks Th1 cell growth by blocking macrophages from making IL-12.
IFN- blocks Th2 cell growth by blocking Th2 from making IL-4.

25. How Do the Actions of Cytokines Affect Immunity?

26. Cytokine Actions
Some cytokines are involved in mediating and regulating innate immunity (Pro-inflammatory cytokines).
Some cytokines are involved in mediating and regulating specific immunity.
Some cytokines are involved in mediating and regulating both innate immunity and specific immunity.

27. Most Important Cytokines

28. Principle Cytokines in Acquired Immunity
IL-2
IL-3
GM-CSF
IFN-
CD8 T cell
IL-1
IL-12
CD4 Th1
Cytolysis:
FAS/FAS ligand
TNF
Granzyme
Perforin M
IL-4
IL-5
IL-6
IL-9
IL10
IL-13
B cell
IL-1
CD4 Th2
Antibody

29. Triggers for Th1 Cell Predomination
Naïve helper T cells are strongly stimulated to become Th1 cells and cell-mediated immunity predominates:
When IL12 production by macrophages is high.
IL-23 and IL-27, relatives of IL-12 that share some similarities to IL-12, also play a role to activate Th1 cells.
When IFN- levels are high.
IL-18, a relative of IL-1 that shares some similarities to IL-1, also activates Th1 cells.

30. Triggers for Th2 Cell Predomination
Naïve helper T cells are strongly stimulated to become Th2 cells and humoral immunity predominates:
When IL-4 levels are high.
When IL-10 levels are high.

31. Who Holds the High Trumps?
If IL-4 levels are low and IL-12 and IFN- levels are high, Th1 cells develop.
If IL-4 levels are high, Th2 cells develop.
Thus, IL-4 holds the high trumps.

32. Is There Cross-Regulation?
If naïve T cells are triggered to become Th1 cells, they produce IFN- which shuts down Th2 proliferation.
If naïve T cells are triggered to become Th2 cells, they produce IL-10 which shuts down macrophage production of IL-12, removing the stimulus for naïve helper T cells to become Th1 cells.
Thus, each local environment establishes its own balance of Th1 and Th2 cells.

33. Principle Cytokines in Acquired Immunity
IL-2
IL-3
GM-CSF
IFN-
CD8 T cell
IL-1
IL-12
CD4 Th1
IFN-
Cytolysis:
FAS/FAS ligand
TNF
Granzyme
Perforin M
IL-4
IL-5
IL-6
IL-9
IL10
IL-13
B cell
IL-1
CD4 Th2
Antibody

34. Treg Cells
Another subset of CD4+ T cells has been identified: Treg cells.
Treg cells produce IL-4, IL-10, and TGF-
Treg cells suppresses T helper cell activity through contact with the T helper cell.
Limits autoimmune T cell activity.

35. Why Is T helper Cell Regulation Important?
Ex., Mycobacterium leprae infection
If Th1 cells predominate, tuberuloid leprosy develops.
Most of the Mycobacteria are walled off in granulomas, with slow tissue destruction and nerve damage caused by a few “free” Mycobacteria.
The patient survives.
If Th2 cells predominate, lepromatous leprosy develops.
The Mycobacteria become widely disseminated in macrophages and the patient has infection of the bone and cartilage, with extensive tissue and nerve damage.
The patient dies.
Different T helper cells are needed to control different diseases.

37. Cytokines Activate Their Target Cells by Binding to Specific Receptors
Cytokines bind to specific receptors on the cell surface.
Cytokines have their signal passed through the cell membrane by changes in receptor configuration.
Cytokines have their signal passed through the cytoplasm by the generation of “second” messengers.
Cytokine activated transcription factors up-regulate and down-regulate transcription.

38. Cytokine Families and the Receptors To Which They Bind
IL-1 family
Ig Domain Receptor Superfamily
Hematopoietin family
Type I Cytokine Receptor Superfamily
Interferon family
Type II Cytokine Receptor Family
TNF family
TNF Receptor Family
Chemokine family
Seven Transmembrane Helix Family

39. Role of the Different Receptor Subunits
Specific subunit ( subunit, low affinity subunit)
Binds specifically to a particular cytokine
Does not transduce a signal through the membrane
Common subunit ( subunit,  subunit, gp130)
Binds to two or more cytokines that are members of the same group, when they are bound to the specific subunit
Transduces a signal through the membrane
Note that the loss of the common subunit eliminates the activity of all cytokines of the family.
Example, loss of common  chain causes loss of IL-2, IL-15, IL-7, IL-9, and IL-4 activities.
Loss of common  chain causes severe combined immunodeficiency disorder (bubble baby)

40. Low Affinity Versus High Affinity Receptors

41. Consequences of Signaling Through a Common Subunit
Explains redundancy:
(1) IL-3 & IL-5 induce
bone marrow proliferation
(2) all three induce
eosinophil proliferation
and basophil degranulation
Note, however, that not all
cells express  subunits
for the three different
cytokines, so there can
still be some specificity.

42. Consequences of Signaling Through a Common Subunit
Because of a limited
number of  subunits,
IL-3 and GM-CSF
exhibit antagonism.
This is particularly
apparent if IL-3 is
added before GM-CSF.

43. How Does a Cytokine Bind to Its Receptor?
Example: The IL-2 receptor is composed of three subunits.
 chain: binds IL-2
 chain: binds IL-2
 chain: signal transducing chain

44. Affinities of IL-2 Receptor Multimers
The strength of
cytokine binding
increases with the
addition of the
common subunits.

45. Note: (1) IL-2 binds to a pocket formed by the  and  subunits
(2) the  subunit binds to the  subunit but not to the  subunit

46. Signal Transduction Across the Cell Membrane
Example: IFN signal transduction.

47. JAK kinases (Jak1, Tyk2) bind to intracellular domain of receptor.
The IFN-/ Receptor
JAK kinases (Jak1, Tyk2) bind
to intracellular domain of
receptor.
JAK kinase phosphorylates
tyrosines on receptor and
itself (autophosphorylation).
Activated JAK kinase
phosphorylates tyrosines on
STATs (signal
transducer/activator of
transcription).
Activated STATs become
active transcription factors.
JAK = Janus kinase because it has two “faces” or functions: binding to cytokine receptor and kinase domain
Others, tired of so many kinases, state that JAK means “just another kinase.”

48. Differences in the Receptors for Different IFNs
Vilcek, J. Nat Immunol Jan;4(1):8-9
Permission for use: Nature Immunology

49. Janus kinases
are used by a
number of class I
and class II
cytokine receptors.

50. Clinical Focus: Therapy with Interferons

51. Rationale for Interferon Therapy
Antiviral agent
Block replication of viruses in viral diseases
Antiproliferative agent
Block replication of cancer cells
Immunomodulatory agent
Enhance macrophage activity
Enhance NK cell activity
Enhance cytotoxic T cell activity

52. Types of Interferons Type I interferons Type II interferon IFN- IFN-
Also, IFN-, IFN-, IFN-, IFN-, IFN-, IFN-
Type II interferon
IFN-

53. Diseases Successfully Treated with IFN-
Hepatitis
Hepatitis B
Hepatitis C
White blood cell cancers
Hairy cell leukemia (now purine nucleoside analogs are better)
Chronic myelogenous leukemia
Solid tumors
Melanoma
Kaposi’s sarcoma
Renal cancer

54. Diseases Successfully Treated with IFN-
Multiple sclerosis
IFN- therapy reduces the number of exacerbations (attacks) and the size of the lesions in multiple sclerosis patients.

55. Diseases Successfully Treated with IFN-
Chronic granulomatous disease
Characteristics of chronic granulomatous disease
Impaired ability of phagocytes to kill ingested microbes.
Suffer recurrent infections
IFN- treatment
Increases ability of phagocytes to produce reactive oxygen and nitrogen molecules
Decreases number and severity of infections

56. Side Effects of IFN Therapy
Bone marrow suppression
Leukopenia
Neutropenia
Anemia
Thrombocytopenia
Hepatic disorders
Renal disorders
Hypertriglyceridemia
Psychiatric considerations
Impaired concentration and decreased alertness
Deficits in verbal memory
Depression