Dr. William Bowers 

Edited and illustrated by Dr Richard Hunt

Medical Microbiology (MBIM 650/720)

SUGGESTED READING: Roitt, Brostoff, Male, 6th Edition, Mosby, 2001  Chapter 8, pp. 132-136; Chapter 7, pp. 119-129

  IMMUNOLOGY - CHAPTER   TWELVE 

  CELL-MEDIATED IMMUNITY: 
  Cell interactions in specific immune responses 

TEACHING OBJECTIVES
Helper T cell-B cell interactions for antibody formation against hapten-conjugated proteins and complex proteins
Thymus-independent antigens
Properties and functions of cytokines

Figure 1

Molecules involved in the interactions of B and TH cells
Antigen is processed by B cell. Co-stimulators are expressed. The processed antigen peptide is presented in association with MHC class II antigens. The T cell recognizes the peptide along with the MHC antigen and the co-stimulators. The T cell expresses CD40 ligand. The latter binds to CD40 antigen on the B cell and the B cells divide and differentiate. Antibodies are produced by the B cell

I. HELPER T CELL-B CELL INTERACTIONS IN ANTIBODY FORMATION

A. Hapten-carrier effect  

Historically one of the major findings was that T cells and B cells are required in order to produce antibody to a complex protein. A major contribution to our understanding of this process came from studies on the formation of anti-hapten antibodies.

Recall that a hapten injected by itself cannot elicit an antibody response. Rather antibodies against haptens require that the hapten be conjugated to a protein (sometimes termed a carrier).

These studies with hapten-carrier established that:

1. Th cells recognize the carrier, and B cells recognize hapten.
2. There must be cooperation between hapten-specific B cells and protein (carrier)-specific helper T cells.
3. Interaction between the hapten-specific B cell and the carrier-specific helper T cell are class II self MHC-restricted. The helper T cell cooperates only with B cells that express class II MHC molecules recognized as self by the T cells.

B. B cells as antigen presenting cells

B cells occupy a unique position in immune responses because they express immunoglobulin (Ig) and class II MHC molecules on their cell surface. They therefore are capable of producing antibody having the same specificity as that expressed by their immunoglobulin receptor; in addition they can function as an antigen presenting cell. In terms of the hapten-carrier protein findings, the mechanism is thought to be the following: the hapten is recognized by the Ig receptor, the hapten-carrier brought into the B cell, processed, and peptide fragments of the carrier protein presented to a helper T cell. Activation of the T cell results in the production of cytokines that enable the hapten-specific B cell to become activated to produce soluble anti-hapten antibodies. Figure 1 summarizes the B cell-T cell interactions that occur.

Note that there are multiple signals delivered to the B cells in this model of Th cell-B cell interaction. As was the case for activation of T cells where the signal derived from the TCR recognition of a peptide-MHC molecule was by itself insufficient for T cell activation, so too for the B cell. Binding of an antigen to the immunoglobulin receptor delivers one signal to the B cell, but that is insufficient. Second signals delivered by costimulatory molecules are required; the most important of these is CD40L on the T cell that binds to CD40 on the B cell to initiate delivery of a second signal.

C. Extension of this model to complex protein antigens (T-dependent antigens)

The same mechanism described above can cover all multideterminant complex protein antigens that require helper T cells. These antigens are referred to as thymus-dependent antigens. If one determinant is recognized by B cells (analogous to the hapten) and the same or different determinant is recognized by the helper T cells (analogous to the carrier), the same model applies. This is shown in Figure 2.

D. B cells in secondary responses

As a consequence of a primary response, many memory B cells are created. These carry a high-affinity receptor, Ig, which allows them to bind and present antigen at much lower concentrations than is required for macrophages or dendritic cells.

II. THYMUS-INDEPENDENT ANTIGENS

The thymus-independent antigens (T-independent antigens) are those that produce normal antibody responses in athymic (thymus-less or nude) mice, i.e. under conditions where T cells are absent. T-independent antigens have the following properties:

1. activate B cells at high concentrations, i.e. are polyclonal B cell activators (antigens like lipopolysaccharide, LPS, sometimes termed B cell mitogens).
2. are large polymeric molecules with repeating antigenic determinants.
3. are particularly resistant to degradation
4. Some antigens activate both immature and mature B cells; other antigens activate only mature B cells and are thus not especially effective in infants where B cells are mostly immature.
5. Responses to several T-independent antigens are dominated by CD5 B cells, described below.

Unlike the thymus-dependent antigens, the thymus-independent antigens:

1. do not produce isotype switching (IgM is almost exclusively produced)
2. do not demonstrate affinity maturation (in which antibodies of progressively higher affinity are produced)
3. do not show secondary responses (no memory B cells).

The thymus-independent antigen pathway is important because humoral immunity is the major mechanism of defense against many harmful bacteria that have polysaccharides in their cell wall. Individuals with depressed T cell systems can still resist these types of bacterial infections.

III. CD5⁺ B CELLS

CD5⁺ B cells (sometimes referred to as B-1 cells) form a population that is distinct from conventional B cells (sometimes referred to as B-2 cells). They have the following characteristics:

1. are the first B cells to appear in ontogeny
2. express surface IgM, but little or no IgD
3. produce immunoglobulins, mainly IgM, from unmutated or minimally mutated germline genes
4. produce antibodies of low avidity that are polyreactive (i.e., bind multiple different antigens, mainly bacterial polysaccharides and double stranded DNA)
5. contribute most of the IgM found in adult serum
6. do not develop into memory cells
7. are self-renewing in adults (i.e., do not continue to arise from a stem cell in the bone marrow as do conventional B cells)
8. reside in peripheral tissues and are the predominant lymphocyte in the peritoneal cavity.

The following table contrasts CD5⁺ B cells with conventional B cells.

As shown in Figure 4, tumors can arise from CD5⁺ B cells and conventional B cells at various stages in their development.

IV. CYTOKINES

Cytokines are a diverse group of non-antibody proteins released by cells that act as intercellular mediators, especially in immune processes.

A. Cytokines are clinically important as biological response modifiers. Terms in the literature:

1. Monokines - produced by mononuclear phagocytes
2. Lymphokines - produced by activated T cells, primarily helper T cells
3. Interleukins - name given to many cytokines, abbreviated as IL and given a number

B. Properties

1. Produced by cells involved in both natural and specific immunity
2. Mediate and regulate immune and inflammatory responses
3. Secretion is brief and limited

a. Cytokines are not stored as pre-formed molecules
b. Synthesis is initiated by new short-lived gene transcription
c. mRNA is short-lived
d. This results in production of cytokine as needed

4. Many individual cytokines are produced by many cell types and act on many cell types (they are pleiotropic)
5. In many cases cytokines have similar actions (they are redundant). Redundancy is due to the following: Receptors for cytokines are heterodimers (sometimes heterotrimers) that can be grouped into families in which one subunit is common to all members of a given family. Some examples are shown in Figure 5.

Since the subunit common to all members of the family functions in binding cytokine and in signal transduction, a receptor for one cytokine can often respond to another cytokine in the same family. Thus, an individual lacking IL-2, for example, is not adversely affected because other cytokines (IL-15, IL-7, IL-9, etc.) assume its function. Similarly, a mutation in a cytokine receptor subunit other than the one in common often has little effect. On the other hand, a mutation in the common subunit has profound effects. Again, as an example, mutation in the gene for the IL-2Rgamma subunit causes human X-linked severe combined immunodeficiency (XSCID) characterized by a complete or nearly complete T cell defect.

6. Often influence the synthesis of other cytokines

a. They can produce cascades, or enhance or suppress production of other cytokines
b. They exert positive or negative regulatory mechanisms for immune and inflammatory responses

7. Often influence the action of other cytokines. Effects can be:

a. antagonistic
b. additive
c. greater than additive (synergistic)

8. Bind to specific receptors on target cells with high affinity. Compare with antigen binding to antibody or peptide binding to a MHC molecule which both show much lower binding affinities.

9. Cells that can respond to a cytokine are:

a. same cell that secreted cytokine: autocrine
b. a nearby cell: paracrine
c. a distant cell reached through the circulation: endocrine

10. Cellular responses to cytokines are generally slow (hours), require new mRNA and protein synthesis

C. Cytokines can be grouped according to function

1. Mediators and regulators of Natural Immunity
     Tumor Necrosis Factor (TNF)
     Interleukin-1 (IL-1)
     Chemokines
     Interleukin-10
     Interferon-gamma (IFN-gamma)

2. Mediators and regulators of specific immunity
     Interleukin-2 (IL-2)
     Interleukin-4 (IL-4)
     Interleukin-5 (IL-5)
     Interleukin-10 (IL-10)
     Interferon-gamma (IFN-gamma)

3. Stimulators of hematopoeisis
     Interleukin-3 (IL-3)
     Colony-Stimulating Factors (CSFs)

D. Functions of selected cytokines: Mediators and regulators of natural immunity

1. Tumor Necrosis Factor (TNF) also called TNF-gamma 

a)   is produced by activated macrophages
b)   is the most important mediator of acute inflammation in response to Gram-negative bacteria and  other infectious microbes
c)   mediates the recruitment of polymorphonuclear leukocytes (PMNs) and monocytes to the  site of infection:     
        i)  stimulates endothelial cells to express new adhesion molecules that make the cell surface "sticky" for PMN and monocytes
        ii)  stimulates endothelial cells and macrophages to produce chemokines that induce leukocyte chemotaxis and recruitment 
d)   acts on the hypothalamus to produce fever
e)   promotes the production of acute phase proteins by the liver

 2. Interleukin-1

a)   produced by activated macrophages
b)   effects are similar to those of TNF

 3. Chemokines

 The name chemokine is a contraction of chemotactic cytokines
 a)   These are are a large family of substances (more than 50) produced by many different leukocytes and tissue cells
 b)   They recruit leukocytes to sites of infection
 c)   They play a role in lymphocyte trafficking

4. Interleukin-10

 a)  is produced by activated macrophages
 b)  acts as an inhibitor of activated macrophages by blocking production of TNF
       

E.   Functions of Selected Cytokines: Mediators and Regulators of Specific Immunity

1. Interleukin-2

a)   is produced mainly by helper T cells (CD4+); less by cytoxic T cells (CD8+)
b)   mainly functions to promote T cell division and to increase production of other cytokines
c)   has other functions that are shown in Figure 6
d)   has autocrine functions on T cell proliferation as depicted in Figure 7

2. Interleukin-4

a)   is produced mainly by Th2 subpopulation of helper T cells (CD4+).  RECALL that Th2 cells are required for antibody production by B cells
b)   stimulates immunoglobulin class switching to the IgE isotype.  (IgE is involved in eosinophil-mediated elimination of helminths and arthropods.)
 c)  stimulates development of Th2 cells from naive CD4+ T cells
 d)  promotes growth of differentiated Th2 cells

3. Interleukin-5

a)   is produced mainly by the Th2 subpopulation of helper T cells (CD4+)
b)   promotes growth and differentiation of eosinophils
c)   activates mature eosinophils

IL-4 and IL-5 function together
IgE opsonizes helminths that then bind to eosinophils which upon activation kill  the helminth.

4. Interferons (IFN)
      
   There are three groups of interferons:  IFN-alpha , IFN-beta , IFN-gamma 

a)   IFN-alpha:  Twenty  variants are produced by leukocytes in response to viruses
        
b)   IFN-beta: This is a single protein produced by fibroblasts and other cells in response to viruses

Both IFN-alpha  and IFN-beta   inhibit viral replication and increase expression of class I MHC on cells

c)   IFN-gamma:
          i)  This protein is produced by  the Th1 subpopulation of helper T cells (CD4+), cytotoxic T cells (CD8+), and NK cells. RECALL that Th1 cells are involved in the elimination of pathogens residing intracellularly in vesicular compartments.
        ii)  IFN-gamma functions in both natural and specific immunity

Natural Immunity
- IFN-gamma enhances the microbicidal function of macrophages through formation of nitric oxide and reactive oxygen intermediates (ROI)

Specific Immunity
- IFN-gamma stimulates the expression of class I and class II MHC molecules and co-stimulatory molecules on antigen presenting cells
- IFN-gamma promotes the differentiation of naive helper T cells into Th1 cells 
- IFN-gamma activates polymorphonuclear leukocytes (PMN) and cytotoxic T cells and increases the cytotoxicity of NK cells.

These functions are shown in Figure 8.       
       
        
 5. Transforming Growth Factor (TGF-beta)

a)   is an inhibitory cytokine produced by T cells, macrophages, and many other cell types.
b)   inhibits proliferation and differentiation of T cells
c)   inhibits activation of macrophages
d)   acts on PMN and endothelial cells to block the effects of pro-inflammatory cytokines

 F.   Functions of Selected Cytokines: Stimulators of Hematopoiesis

1. Interleukin-3

a)   produced by helper T cells
b)   promotes growth and differentiation of bone marrow progenitors

 2. Colony-Stimulating Factors (CSFs)

a)   produced by T cells, macrophages, endothelial cells, fibroblasts
b)   granulocyte-macrophage colony-stimulating factor (GM-CSF) promotes growth and
differentiation of bone marrow progenitors
c)   macrophage colony-stimulating factor (M-CSF) is involved in the development and
function of monocytes/macrophages
d)   granulocyte colony-stimulatory factor (G-CSF) stimulates the production of PMN

G.   Cytokine Network
 Although the focus has been on the production and action of cytokines on cells of the immune system, it is important to understand that many of them have effects on other cells and organ systems.  A schematic diagram showing some of the interactions in the cytokine network is presented in Figure 9.

Figure 9a
Cytokine network. Communication between lymphocytes and macrophages and other components of the immune system

Figure 9b  Cytokine network. Communication between lymphocytes and macrophages and the hypothalamus, adrenals and the liver

Figure 9c Cytokine network. Communication between lymphocytes and macrophages and other cells and tissues

Table 2  -  FEATURES OF CYTOKINES
Cytokine	Cell Source	Cell Target	Primary Effects
IL-1	Monocytes  Macrophages Fibroblasts Epithelial cells Endothelial cells Astrocytes	T cells; B cells Endothelial cells Hypothalamus Liver	Costimulatory molecule Activation (inflammation) Fever Acute phase reactants
IL-2	T cells; NK cells	T cells B cells Monocytes	Growth Growth Activation
IL-3	T cells	Bone marrow progenitors	Growth and differentiation
IL-4	T cells	Naive T cells T cells B cells	Differentiation into a TH 2 cell Growth Activation and growth; Isotype switching to IgE
IL-5	T cells	B cells Eosinophils	Growth and activation
IL-6	T cells; Macrophages; Fibroblasts	T cells; B cells Mature B cells Liver	Costimulatory molecule Growth (in humans) Acute phase reactants
IL-8 family	Macrophages; Epithelial cells; Platelets	Neutrophils	Activation and chemotaxis
IL-10	T cells (TH2)	Macrophages T cells	Inhibits APC activity Inhibits cytokine production
IL-12	Macrophages; NK cells	Naive T cells	Differentiation into a TH 1 cell
IFN-gamma	T cells; NK cells	Monocytes Endothelial cells Many tissue cells -  especially macrophages	Activation Activation Increased class I and II MHC
TGF-beta	T cells; Macrophages	T cells Macrophages	Inhibits activation and growth Inhibits activation
GM-CSF	T cells; Macrophages; Endothelial cells, Fibroblasts	Bone marrow progenitors	Growth and differentiation
TNF-alpha	Macrophages; T cells	Similar to IL-1	Similar to IL-1
IL = interleukin GM-CSF = granulocyte-macrophage colony stimulating factor IFN = interferon TNF = tumor necrosis factor TGF = transforming growth factor

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