the major players in adaptive immunity

455RESONANCE � May 2009

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The Major Players inAdaptive Immunity1.Humoral Immunity

Asma Ahmed1, Banishree Saha2, Anand Patwardhan3,Shwetha Shivprasad4 and Dipankar Nandi5

How do we remain healthy, for the most parts, in the midstof an environment teeming with opportunistic and infec-tious microbes, potential carcinogens and allergens? Thefact is that our immune system, by and large, does a fine jobin protecting us. It is therefore important to understand theorganization of the immune network, which is broadly cat-egorized into two groups: innate and adaptive. Cells in-volved in innate immunity are the first to come into contactwith invading microbes, similar to the border security force,and respond rapidly but in a non-specific manner. On theother hand, the cells involved in adaptive immunity areslower to respond but act in a very specific manner. Thoughthe primary response is slow, the secondary response ismuch faster and demonstrates memory. This article willfocus on some important features and key players involvedin the adaptive immune response. The first part deals withthe humoral immune response mediatedmainly by immuno-globulins produced by the B cells. The second part dealswith T cells, the Major Histocompatibility Complex (MHC)-encoded molecules, and Recombination Activating Genes(RAG) responsible for generating diverse B-cell receptors(BCR) and T-cell receptors (TCR). With the advent ofnewer and smarter infectious agents, it is important tounderstand the working of the immune network as moreresearch in this area may facilitate the development ofbetter protective strategies.

The immune system is responsible for protecting us againstmicrobial infections and the spontaneous generationof tumours. Ifthe immunesystemis compromised, residentmicrobesgain advan-

1Asma Ahmed and2Banishree Saha areresearch scholars inDipankar Nandi’s

laboratory.3Anand Patwardhan is afinal year MSc (Biotech-nology) student inShantiniketan, WB.

4Shwetha Shivaprasad isa KVPY scholar and afirst year MSc (Biotech-nology) student at theMS University, Baroda.5Dipankar Nandi is anAssociate Professor inthe Department ofBiochemistry, IISc,

Bangalore.

�

KeywordsImmunity, immunoglobulins, im-munodeficiency, B-cell recep-tor, recombinase, antibody di-versity.

456 RESONANCE � May 2009

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tage, which may be deleterious to the host. An example of this isevident in patients suffering from acquired immune deficiencysyndrome (AIDS)1 who show high susceptibility to opportunisticinfections leading to greatermortality. Another example is that ofDavid Vetter who suffered from congenital immunodeficiency(lack or reduced numbers of immune protective cells or mecha-nisms) and spent his entire life (thirteen years) inside a sterileplastic bubble; hence, the name ‘Bubble boy’.

How does the immune system work? Broadly, its functiondepends on three ingredients: cells, receptors and effector mol-ecules. Similar to a nation’s defence organization, the immunesystem possesses different cells which play distinct roles duringthe immune response (Figure 1). Immune cells possess key cell-surface receptors that recognize microbial constituents such aslipopolysaccharides (LPS) and flagellin, present on the bacterialcell surface. The immune systemcan distinguish between self andnon-self andmounta response byrecognizing the uniquemolecularsignatures present on invadingmicrobes, tumours or foreign cells.Activation of specific receptors on immune cells results in signal-ling events that culminate in the production of various effectormolecules such as free radicals, cytokines, and immunoglobulinsand lead to the eliminationof invadingmicrobes, tumoursor foreigncells.As thedoubling timeofmicrobes is short,multiple strategiesare required to generate a rapid and effective immune response.

1 Acquired Immune DeficienceySyndrome (AIDS): A set of syn-dromes caused by infection withhuman immunodeficiency virus(HIV). The numbers of CD4+ Tcells decrease as the infectionprogresses, resulting in en-hanced susceptibility to oppor-tunistic infections, tumours andother complications.

Figure 1. Cells of the im-mune system. Pluripotentstem cells give rise to theimmunesystem,whichmaybe classified into twogroups: innate and adap-tive. The left-hand sideshows cells involved in in-nate immunity,whichactasthe first line of defence:granulocytes (neutrophils,basophilsandeosoniphils),monocytes and macroph-ages. The right-hand sideshows cells involved inadaptive immunity: B cellssecretespecific IgwhereasT cells are responsible forcell- mediated immune re-sponses. There is plenty ofcross talk between cellsinvolvedinbothformsoftheresponse, e.g., macroph-ages process antigens andpresent peptide/MHC mol-ecules to T cells to initiatethe adaptive immune re-sponse. T cells, upon acti-vation, secrete cytokines,e.g., IFN��, which stimulatemacrophage functions.(See text for details)


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There are two types of immune responses: innate or inborn andadaptive.

The Innate Immune Response

The components of the innate system are responsible for the firstline ofdefence and include anatomic, physiologic, phagocytic andinflammatorybarriers.This response is quickbut lacks specificity.Duringinfections, thenumbersofbloodcellsknownasneutrophils2a

increase and physicians often use this information obtained fromblood tests for confirmatory diagnosis. Increase in the numbers ofeosinophils2b is evident in parasitic helminthic infections, e.g.,infectionwith Ascaris, a parasitic roundwormwhich resides in thesmall intestine. In addition, eosinophils and basophils2calso accu-mulate at inflammatory sites during allergic reactions induced byexternal agents. Cells belonging to the monocyte/macrophage3

lineage ingest and kill microbes. They are also important inprocessing and presenting antigens4 (substances from invadingmicrobes or tumour cells or allergens that elicit an immuneresponse) to T cells to initiate the adaptive immune response,which is discussed below.

Adaptive Immunity

The adaptive immune response ismuch slower but is characterisedby specificity and memory. In fact, these two characteristics areimportant hallmarks of this response. Lymphocytes,which consti-tute 20–40%ofwhite blood cells or leukocytes, are themajor cellsresponsible for adaptive immunity. These are further grouped intoB and T lymphocytes (Figure 1). B cells are responsible forhumoral (present in fluids) immunity whereas T cells mediatecellular immunity. B cells possess B-cell receptors (BCR), alsoknown asmembrane immunoglobulins, on the cell surfacewhich,uponbinding toantigen, result indifferentiationofBcells toplasmacells. These plasma cells secrete copious amounts of solubleimmunoglobulins (or Ig, also known as antibodies) which bind toantigens. Therefore, the active ingredient here, i.e., antibodies, canbe separated awayfromcellswithout any loss in functional activity

2a Neutrophils: Cells of the in-nate immunesystem that usuallyare the first tomigrate to a site ofinfection. Their primary functionis to phagocytose and kill patho-gens2bEosinophils: are a class ofgranulocytes with a bilobednucleus and cytoplasm whichstains with the acidic dye, eosin.They are part of the anti-micro-bial innate defense network andare also involved in allergic re-actions together with basophilsand mast cells. An abnormallyenhancednumber of bloodeosi-nophils, known as eosinophilia,iis indicative of infections, usu-ally parasitic.2cBasophils: are non-phogocyticgranulocytes which form <1%of the total circulatingwhitebloodcell population. Their nuclei arelobedand their cytoplasm stainswith a basic dye, methyuleneblue. Basophils and mast cellsare closely related and are themain mediators of the allergicresponse. they store histamineand release it during an allergicreaction when their Fc recep-tors are cross-linked by aller-gen coated IgE antibodies.3Macrophages : Cells of the in-nate immune system whosemajor functions are phagocyto-sis, antigen processing and pre-sentation.4 Antigen Presenting Cells: Aclass of immune cells which in-cludes dendritic cells, macroph-ages and B cells. They digestcellular ormicrobial-derivedpro-teins to peptides and presentthem in complex with MHC to Tcells.


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(defined as the capacity to bind antigen). Blood serum (liquidcomponentofbloodwithoutanyclottingfactors)containspolyclonalantibodies which are secreted by plasma cells with multiplespecificities.As serumantibodies are produceddue to stimulationby antigens, the presence or absence of specific antibodies has apotential clinical value. Diagnostic kits have been developed todetect antigen-specific antibodies and are used to diagnosemicro-bial infections. Those infected with Salmonella possess highamounts of serumantibodies that clumpor agglutinateSalmonellaantigens and this test is knownas theWidal test,which is oftenusedin the diagnosis of typhoid. Similarly, the presence of antibodiesagainst HIV proteins is the basis for diagnosing HIV infections.

For cellular immunitymediated byT-lymphocytes, cell–cell con-tact is required. T cells express the T-cell receptor (TCR) whichrecognises peptides (short segments of amino acids derived fromself proteins, microbial proteins, tumour5-specific proteinsor selfproteinsmodifiedbybinding to allergens) onpresentingmoleculesknown as the major histocompatibility complex (MHC)-encodedproteins located on the cell surface of antigen-presenting cells(APC). In addition, there is a separate group of lymphocytesknown as natural killer (NK)6 cells which are the key effectors of‘antibody-dependent cell-mediated cytotoxicity’ (ADCC)7 andare important for immunity against tumours. NK receptors do notundergo rearrangement.

How is the antigenic specificity of the immune response achieved?B and T cells express on their surface, receptor proteins knownas BCR or TCRwhich determine antigenic specificity. Each B orT cell expresses a unique BCR or TCR (a heterodimer of twoproteins, either�� or �� ); the total number ofmolecules that theycan recognize is very large as the numberof different combinationspossible are large. This concept that each lymphocyte expressesa unique cell surface receptor for antigen recognition is importantas it allowsonly selected cells containing the appropriate receptorsto proliferate and mount responses during an immune reaction. FMcFarlane Burnet was the first to enunciate this concept known

5 Tumour: Uncontrolled growthof cells due to loss in cell cycleregulation. Plasma cells thatbecome tumorigenic are knownas myeloma.

6 Natural Killer (NK): Lympho-cytes that do not express B cellor T cell antigen receptors butcan kill tumour cells and partici-pate in ADCC.

7 Antibody-Dependent Cell-me-diated Cytotoxicity (ADCC) : Aprocessbywhichantibodyboundinfectedcells are recognizedandkilled by host NK cells, mac-rophages, neutrophils or eosi-nophils which possess recep-tors for lg.


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as the ‘clonal selection hypothesis’ for which he was awarded theNobel Prize in1960 [1]. This concept haspractical applications tooand has led to the development of the ‘hybridoma technology’which enables the isolation and purification of antibodies withunique specificity known as ‘monoclonal antibodies’8. We willexamine lymphocytes and important associatedmolecules neces-sary for their (lymphocytes) optimal function in greater detailbelow.

The Humoral Response

B cells derive their name from ‘Bursa of fabricius’, the organ inbirds in which B cells mature. This was first demonstrated by theobservation that the removal of the Bursa in newly hatchedchickens impairs their ability to generate a humoral response, i.e.,produce antibodies against a specific antigen [2]. Later it wasdiscovered that Bcells originate andmature in the bonemarrowofmice and humans. EachBcell expresses a unique antigen-bindingreceptor called theBCRon its cell surface. CirculatingBcells thathave not been exposed to antigens are known as naïve B cells9,whichget activateduponbindingof theirBCRtoa cognate antigen.B cells internalize antigens, digest them and present peptidesderived from these antigens together with MHC molecules (seebelow) to T cells. Activated T cells induce the expression of cellsurface proteins (e.g., CD40L) and secrete factors known ascytokines which stimulate the activated B cells to divide rapidlyand differentiate intomemoryBcells and plasma cells (Figure 2).This process occurs in specialized areas in lymph nodes or spleenknown as germinal centres. Increase in the numbers of germinalcentres in lymph nodes and spleen correlates with an activehumoral response. Plasmacells are themain effectorBcellswhichsecrete large numbers of immunoglobulin molecules (some esti-mates have it as more than 2000 Ig/second). The first immunoglo-bulin to be secreted in this response is always IgM; subsequently,different cytokines instruct B cells to secrete different classes ofIg, i.e., IgG, IgA and IgE, with the same antigenic specificity (seeFigure 2).Memory cells have a longer life span and they respondquickly upon a second exposure to the same antigen.

8 Monoclonal Antibody: Origi-nating from a single cell and allcells derived from this originalclone possess the identical an-tigenic specificity.

9Naïve Cell: Mature T or B cellsthat have not encountered anti-gen.


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What are Immunoglobulins?

In general, immunoglobulins or antibodies have a similar overallstructure consisting of two polypeptides: heavy chain (highermolecularmass) and light chain (lowermolecularmass).A typicalantibody molecule is ‘Y’ shaped and is composed of two heavychains and two light chains (IgD, IgE and IgG) that are heldtogether by covalent (disulphide bonds) and non-covalent forces,giving rise to two antigen binding sites (Figure 3). G MEdelmanand R R Porter were awarded the Nobel Prize in 1972 for theirpioneering studies that led to the unravellingof the structure of theantibody molecule. Secretory IgA is dimeric (consisting of fourheavy chains and four light chains) whereas IgM is pentameric(consistingof tenheavy chains and ten light chains). There are twotypes of light chains: � (kappa) and � (lambda) and five types ofheavy chains: � (alpha), � (delta), (epsilon), � (gamma), and (mu). A single Ig molecule possesses one type of heavy chainassociatedwith either light chain but never both.Human immuno-globulins are divided into five classes of antibody known asisotypes (Box 1): IgA (�), IgD (�), IgE (), IgG (�), and IgM (),depending on the type of heavy chain which has been denotedwithin brackets. IgG is present in maximal amounts in serumfollowed by IgMand IgAwhereas the amounts of IgD and IgE are

Figure 2.Overviewof cellu-lar events during the gen-eration of the humoral re-sponse. A naïve B cell rec-ognizes antigen and thisbindingdeliversthefirstsig-nal. Also, B cells processantigens and present pep-tides with MHC moleculesto CD4+ T cells. Conse-quently,Tcellsgetactivatedand express the cell sur-facemoleculeCD40Lwhichbinds to CD40 on B cells,thus delivering the secondsignal. These signals, to-gether with cytokines pro-duced by activated T cells,play important roles: IL-2,IL-4 and IL-5 are importantfor proliferation whereasIFN-�� and TGF-� are impor-tant for differentiation ofactivated B cells to plasmacells and memory B cells.Initially, IgM is secreted byplasma cells; subsequen-tly, isotype switching oc-curs under the influence ofdifferent cytokines, result-ing in secretion of differentIg isotypessuchasIgA, IgGand IgE.


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low. IgG and IgA are further classified into subclasses dependingonminor amino acid differences. IgG is subdivided into IgG1, IgG2,IgG3, IgG4 based on minor chain differences and numberedaccording to decreasing serum concentration. IgA is subdividedinto IgA1 and IgA2. IgA1 is amonomer and forms themajor serumIgA while IgA2 is a dimer and the main component of secretoryIgA.

Eachheavyand light chain can be further divided into constant andvariable regions.The first 110 aminoacidsofdifferent immunoglo-bulins show sequence variability (hence the name) and form theantigen-bindingpocket.This characteristic is important as it allowsflexibility in binding to different antigens. Multiple alignment ofamino acid sequences from different immunoglobulins demon-strate that three regions within the variable region show greatervariability and are termed as ‘complementarity determining re-gions’ (CDR), out of which CDR3 shows the greatest variability.On the other hand, there is not much variability in the ‘constantregion’ of antibody molecules. This region contains the site forcarbohydrate addition, i.e., glycosylation and can bind to Fcreceptors on a number of cell types such as NK cells and directdifferent biological activities (Box 1).

What are the Mechanisms Involved in GeneratingAntibody Diversity?

For a long time, immunologists were puzzled by the ability of theimmune system to generate the vast array of antibodies against alarge number of antigens – it would appear that this potential is

Figure3.Structureofa typi-cal Ig molecule. In mostcases, Ig are heterodimersof two heavy and two lightchains.Thenumberofanti-gen binding sites can varyfrom two (e.g. IgA, IgD, IgEand IgG,) to four (e.g.secre-tory IgA) to ten (e.g. IgM).The aminoterminalsofbothheavy and light chains arevariableandareresponsiblefor antigen binding (Fab).The rest of the chains re-mainconstantandform theFc fragment, which is re-sponsible for Ig-mediatedeffector functions, e.g.opsonization and comple-mentactivation (Seetext fordetails).


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Box 1. Different Human Immunoglobulins and their Properties

Class Subclass Molecular Major biological functions(heavy formchain)

IgG(�� IgG1 Monomer Placental transfer to protect the fetus.IgG3 Can activate complement system.IgG4 Opsonization.

IgG1 and IgG3 are often produced against protein antigens whereas IgG2 isproduced against capsular polysaccharides present in some bacteria.In case of deficiency of any of the 4 subclasses, there is increased risk ofasthma or infections.

IgA(�) IgA1 Monomer Found in external secretions, e.g. breast milk, saliva, tears and mucus.IgA2 Dimer is Protects new born babies from infections.

Secretory Important line of mucosal defence against infections.Some individuals have complete absence of IgA, but normal levelsof other Ig. This is called as selective IgA deficiency which is acommon immunodeficiency disease.The majority of these cases are asymptomatic although in some casescases there are increased incidences of infections, autoimmunity andallergy.

IgM() – Pentamer First class of Ig to be produced during a primaryimmune response.Contains 10 antigen binding sites.Activates complement system more efficiently than IgG.Deficiency results in susceptibility to infections, especially by encapsulated bacteria (e.g. Streptococcus pneumoniae etc.) and increased autoimmune disorders, cancer etc.Inability of IgM to switch to other classes causes hyper IgM syndrome withelevated levels of IgM and reduced levels of other Ig subtypes.

IgE() – Monomer Responsible for allergic reactions.Binds to Fc receptor and induces mast cell degranulation.Serum IgE levels increase upon parasitic helminth infections or allergy.Low IgE levels are often associated with immunodeficiency associatedwith low amounts of other Ig isotypes.Hyper IgE syndrome is characterized by recurrent Staphylococcal infections, skin and pulmonary abscesses and increased numbers of bloodeosinophils.

IgD(� ) – Monomer It is present in very low amounts is serum and not much is know of itsroles.IgD and IgM are membrane bound Igs on naïve B cells.IgD deficiency individuals do not show any obvious phenotype.


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infinite! S Tonegawa’s group (Nobel laureate 1987) showed forthe first time that the immunoglobulingenes rearrangeduringBcelldevelopment. In the germ-line DNA there are multiple genesegments that encode a portion of an immunoglobulin’s heavy orlight chain. The genes encoding these chains are arranged as‘multi-gene families’ situated on different chromosomes. Theseregions contain coding sequences that are separated by non-coding regions which are brought together after gene rearrange-ment [3]. The light chain family contains ~76 variable genes (V)and 5 joining genes (J) and a unique constant (C) gene (Chromo-some 2p). There are ~73–74 Vl genes, 7–11 Jl and 7–11 Cl genesthatmayencode the human light chain (Chromosome22q).On theother hand, there are 123–129 VH, 27 diversity (D) and 9 JH genesand 11 CH genes that can encode the human Ig heavy chain(Chromosome 14q). Hence, the ability of any of the VH genesegments to combinewith anyof the 27DH gene segments and anyof the 9 JH segments and 11 CH genes gives rise to large numbersof possible combinations (V � D � J �C). Similarly for � and �there will also be large numbers of combinations, and this mecha-nismallows humans to synthesizemillions of different types of Ig.

This recombination of V, D and J coding genes of B- and T-cellreceptors are performed by enzymes collectively called ‘VDJrecombinase’. The products of two genes known as Recombina-tionActivatingGene (RAG)-1 andRAG-2 form this recombinase(Figure 4). Apart fromVDJ recombination, several other mecha-nisms have been identified in humans that generate additionalantibodydiversity.These include junctional flexibility,P-nucleotideaddition (occurs due to variation in endonucleatic cleavage result-ing in addition of nucleotides to formapalindromic sequence), N-nucleotide addition (addition of nucleotides in the free 3’end ofDNA by terminal deoxynucleotidyl transferase), class switchingand combinatorial association of light and heavy chains. Theseprocesses result in expressionof immunoglobulinmolecules on theB-cell surface.

Furthermodifications contributing to IgdiversityoccuruponBcell

This recombinationof V, D and J codinggenes of B- and T-cell receptors areperformedbyenzymes collectivelycalled ‘VDJrecombinase’.


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activation. For example, it is well known that IgM is on theB-cellsurface but is secreted by plasma cells. This process involvesRNAsplicing and the heavy chains of secreted Igpossess a stretchof 20 hydrophilic amino acids at the C-terminus, whereas mem-brane Ig possess a stretch of 40 amino acids comprised of ahydrophilic segment followed by a hydrophobic transmembrane

Figure 4. Overview of RAG mediated Ig gene rearrangement. Germline DNA contains three Igmultigene families: �� light chain, � light chain and the heavy chain, which are located on differentchromosomes. During B cell maturation, gene segments of these families are rearranged to yieldfunctional Ig molecules. The light chain families possess V, J and C segments whereas the heavychain family has V, D, J and C segments. With the help of RAG proteins V, D and J segments arerearranged to give rise to the variable region of the heavy and light chains. Rearrangement of V, Dand J segments in different permutations and combinations generates Ig diversity. The rearrangedvariablesegments then join theconstant region fragments toproduce the finalheavyand lightchaintranscripts. These are translated to form the heavy and light chain proteinswhich assemble to forma functional Igmolecule.

Germ-lineDNA

VL1 VL2 ….VL40DH1…..DH27 JH1… JH6 Constant μ JL1…. JL6 C? \?

Rearranged DNA in B-cell

Primary mRNA transcript

mRNA

Nascent polypeptideLight chainprocessing

Heavy chainprocessing

Assembly into a

Complete Antibody molecule

DNA

RNA

Protein

AAA(n) AAA(n)

VJ recombinationRAG 1 and 2 + other proteins

Transcription Transcription

VDJ recombination by

splicingsplicing

Translation Translation

VH1 VH2 ….VH51

V D J V J

Dimerization

VVVH1HVVVVH1HVVVVH1HVVV 1VVVH1HVH1HVVVVVH1HVV 1VVVH1HVH1HVVH1HVVVVVVH1HHVVH1HVVVH1HHVVVVH1HHVVHHHHHHH VHVV 2VVVVHVV 22VVHVV 2VVHVV 2VHVV 2VVHVV 2VVVHVV 2VHVV 2VHVV 2VVVHVV 2VVVHVV 2V 2VHVV 2VVHVV 2VVHVV 2VVHVVVHVV 2VVHHHHHHHHHHHHH 5.VHV 5VVHV 5.V 5VVVVHVVHV 5VVVHVVHVVVHVVVHVVHVVVVHVVHVVVHVVHVVHVVHVVVHHHHHHHHH DHDDDHDHDHDDHDHDHDHHHHHHHHHHHHHHHH …..……..…..……...………….. HH27277272 JJHJJJJ HH μμμμμμμμμμμ VVV 1VV 1VVVVVV 1VVVVVV 1V 1VVL1LVVVVVVVVL1LVL1LVL1LVVLL1LL1LL1LLLLLLLL1LLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLL V 2VVVV 2VVV 2VV 2VVVVVVLVV 2VLVV 2VVVLVV 2V 2VVVVVLVV 2VVLVV 2VLVV 2V 2VVLVVLVV 2VLLLLLLLLLLLLLLLLLLLLLLLLLLLLLL 0V 4VV 4VVV 44VV 4V 4VVL4VL4VVVVL44VVVL4VVVVL4VVL4V 4VVLVVL4VL4VVVL4VLLLLLLLLLLLLLLLLLL LLL CC?? \CCC?? \?? \?? \?? \?? \???? \????? \???? \?????? \????? \???? \??? \?????C \C \\\\\\\\\\\\CC??????? \? \?????? \\\\\\\\\??????? \? \??????? \\\\\\\\??????? \? \??????? \\\\\\\\\\???? \? \?????? \\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\Germ-lineDNA

VL1 VL2 ….VL40DH1…..DH27 JH1… JH6 Constant μ JL1…. JL6 C? \?

JJJJJJJJJJVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVV JJJJJJJJJRearranged DNA in B-cell

Primary mRNA transcript

mRNA

Nascent polypeptideLight chainprocessing

Heavy chainprocessing

Assembly into a

Complete Antibody molecule

DNA

RNA

Protein

AAA(n) AAA(n)

VJ recombinationRAG 1 and 2 + other proteins

Transcription Transcription

VDJ recombination by

splicingsplicing

Translation Translation

VH1 VH2 ….VH51

V D J V J

Dimerization


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domainanda short hydrophilic cytoplasmicportion.Thehydropho-bic segment is encoded by two exons, M1 andM2, present in theheavy chain encodingDNAof all immunoglobulin isotypes. If themRNAtranscript is spliced such thatM1andM2are included, thenthe translatedprotein is amembrane immunoglobulin.However, ifM1andM2are excludedduring splicing then the immunoglobulinmolecules are secreted. Most likely, the process of activation anddifferentiation into plasma cells directs altered splicing of themRNA transcript.

IgM is the first isotype to be secreted by plasma cells and, withtime, other isotypes with the same antigenic specificity appear incirculation. During this process, the variable region remains thesame (hence there is no alteration in antigenic specificity)whereasthe constant region of the heavy chain of the immunoglobulinchanges (known as ‘isotype switching’). The switch to otherisotypes is determined by the cytokines secreted by the TH cells.For example, Interleukin10 (IL)-4 induces switching to IgG1 andIgE whereas IL-5 induces switching to IgA. DNA sequencesknown as ‘switch sites’ are located upstream of the heavy chainconstant regions. The order of heavy chain constant regions in theDNA is: IgM, IgD, IgG, IgE followed by IgA. IgMand IgD do nothave any upstream switch sites and are the first isotypes to beexpressed on the cell surface of naïve B cells. If a switch occursfrom IgM to IgE, then the DNA sequence from IgM to IgG islooped and excised and the functional transcript will now containthe heavy chain constant region of IgE.

During B-cell response it is often found that antibodies producedearly during the immune response bind to the antigen with lowaffinity whereas those produced later bind with stronger affinity.This process is known as ‘affinitymaturation’ and themechanisminvolves ‘somatic hypermutation’.Randommutations occur in thevariable regions of immunoglobulins of individual B cells thatenhance the binding of the antibodywith its cognate antigen. Thisprocess results in the selection and secretion of high-affinityantibodies by plasma cells.

10 Interleukins: A group of lowmolecular proteins secreted bya variety of cells that regulatedifferent immune functions: pro-liferation, death, differentiation,antibody secretion, etc. IL-2 in-creases proliferation of T cellswhereas IL-4 regulates IgE pro-duction by B cells.


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In summary, rearrangement of immunoglobulin genes results in thegeneration of large numbers ofB cellswith differentmembrane Igmolecules. Upon binding to an antigen, B cells are triggered,followed by secretion of antibodies and isotype switching andbinding affinities are increased further by somatic hypermutation.Thesemultiple strategies, i.e., “first bind, adapt and, subsequently,bindwith greater affinity” allowsgreater flexibility to the immunesystem to generate specific antibodies against a limitless numberof antigens.

Antibodies Play Different Effector Roles

Thevariable regionsof antibodiesbind toantigens vianon-covalentinteractions, e.g., hydrogen bonds, ionic bonds, hydrophobic inter-actions and van der Waals interactions. It is this binding thatneutralizes toxins and viruses rendering them biologically non-functional. Attenuated viruses (e.g., small pox, polio) or toxoids(inactivated toxins, e.g., tetanus exotoxin, diphtheria exotoxin) areused to elicit specific antibodies andmemory cells by the host viaa processknownas‘active immunization’. In essence, vaccinationallows the host to produce high amounts of neutralizingantibodiesthat bind to pathogens and render them non-functional (i.e., theyare not capable of replicating) and prepare the host to counter apossible infection. In fact themost effective vaccines are ones thatgenerate good neutralizing antibodies. Sometimes, preformedantibodies (for example from horse or goat) are administeredagainst rapidly acting toxins or snake/insect bites. Such ‘passiveimmunization’is helpful in conditionswhen there is notmuch timefor the host to produce antibodies and neutralize toxins (e.g.,botunilum toxin) or snake venoms. Anti-snake venoms containimmunoglobulins that will neutralize venoms of many commonpoisonous snakes found in India, e.g., cobra, kraits andvipers. Thisaspect is important as there is no time to ascertain the identity ofthe snake that has bitten the patient and quick therapy is a must insuch cases.

Complement Activation: The antigen-antibody complexes acti-vate the complement11 system, which consists of several plasma


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proteins that are present in blood in an inactive state. Onceactivated, they clear pathogens by cell lysis. Some antibodies bindtomicrobes and enhance their phagocytosis (this process is knownas opsonization) by promoting adhesion to neutrophils and mac-rophages. Some antibody-coated target cells are lysedbyNKcellsby ADCC, as described earlier. Here the constant region of theantibody is bound to CD1612 receptors on NK cells that getactivated and attack antibody-bound cells leading to their lysis.

Protection ofMucosal Surfaces: Some Ighave specialized roles,e.g., secretory IgA is the major class of Ig present in mucosalsecretions (tears, saliva, intestinal secretions, etc.) andplays a keyrole in protecting such environments in the body. Themechanismby which secretory IgA is present in mucosal secretions is due toan interesting cell biological process. Plasma cells secrete IgAwhich is recognized by polymeric Ig receptor (pIgR) present inepithelial cells. These receptors present on the basolateral surfacebind to IgA and transport these molecules to the luminal surface.Here, there is cleavage of the receptor and IgA is released in thelumen togetherwith a remnant of the pIgRknownas the ‘secretorycomponent’.

Ig and allergy: An immunoglobulin isotype known as IgE isimportant for allergic responses. IgE binds to specific receptorspresent on themembranes of tissuemast cells and bloodbasophils.Once an allergen cross-links a receptor-bound IgE on cells, itinduces degranulation, resulting in release of several effectormolecules, including histamines13, leukotrienes, etc. These com-pounds lead to dilation of blood vessels, increasedmucous secre-tions (runnynose) andmore severe effects leading todrop in bloodpressure, collapse of the respiratory, circulatory systems, etc.

Immunoglobulins andAutoimmunity: In some cases, antibodiesare formed against the host, leading to autoimmunity (Box 2).Rheumatoid arthritis is one such autoimmune disorder, whereantibodies are formed against the Fc region of IgG. Patientssuffering from rheumatoid arthritis possess high amounts of anIgMknown as rheumatoid factor. It binds to the circulating IgG, to

11 Complement: A number ofserum proteins which, uponactivitation, cause lysis of anti-body bound infected cells orpathogens.

12Cluster of Differentiation (CD) :Cell surface molecules usuallypresent on leukocytes that areidentified using monoclonal an-tibodies, e.g. CD3 is present onT cells whereas CD19 is presenton B cells.

13 Histamines: Derivatives ofthe amino acidHistidine that arereleased by mast cells upon ex-posure to allergens. Binding totheir receptors leads to smoothmuscle contraction, mucus se-cretion, etc. Anti-histamines arewidely used to counter the ef-fects of allergy.


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form IgM–IgG complexes, which get deposited in joints causinginflammation.Antibodies against selfmolecules are also producedinotherautoimmunediseases, e.g., SystemicLupusErythromatosusand Graves disease (Box 2).

Box 2. A List of Some Autoimmune Disorders

Disorder Description

Insulin Dependent Diabetes Develops due to autoreactive T cells attacking and destroyingMellitus (IDDM) insulin secreting beta cells in the pancreas. Eventually, the body’s

insulin production drops and blood glucose levels rise. The mostcommon therapy used is administration of insulin. This disease affects~0.2% of the world’s population.

Graves’ disease People suffering from this disease produce auto-Ig to the thyroid-stimulating hormone (TSH) receptor. Binding of these Ig to thereceptor results in overproduction of thyroid hormones, thyroxineand triiodothyronine, in afflicted individuals.

Myasthenia Gravis Patients suffering from myasthenia gravis produce auto-Ig, whichbind to acetylcholine receptors on muscle cells, and block the bindingof acetylcholine. This leads to the inhibition of neurotransmittersignaling and destruction of antibody bound muscle cells due tocomplement activation. Eventually, there is weakening of skeletalmuscles (muscle fatigue) and it is a struggle to perform normalchores, e.g. eating.

Systemic Lupus Erythromatosus A systemic disorder where an autoimmune response is(SLE) generated against awiderangeofantigens andseveral organs are affected.

Affected individuals produce auto-Ig to DNA, histones, etc.and antigen-Ig complexes lead to hemolytic anemia, glomerulonephritis etc. Women are affected ten times more by SLE than men andit develops between the ages of 20 – 40. One of the hallmark featuresof SLE is the appearance of a “butterfly” rash on the face.

Multiple Sclerosis It is one of the most common causes of neurologic disability.Autoreactive T cells form lesions along the myelin sheath of nervefibres leading to numbness and paralysis of limbs.

Rheumatoid arthritis It is a fairly common autoimmune disease and arises due toproduction of auto-Ig (mainly IgM) reactive to Fc regions ofIgG. IgM-IgG complexes deposit in bone joints and triggerinflammation.


GENERAL � ARTICLE

Monoclonal Antibodies: As mentioned previously, serum Ig arepolyclonal as theyoriginate fromdifferent plasma cells.However,as the original B cell is clonal in origin, it is possible to isolate andpropagate such clones using the hybridoma technologydevelopedbyGKohler andCMilstein (Nobel Prize, 1984).The advantage isthatB-cell hybridsgrowcontinuously in culture andproduce Ig thatareof single antigenic specificity, knownasmonoclonal antibodies.It involves the fusion of antibody-producing plasma cells (Igsecretion)withanon-Igsecretingmyelomacell line (ability to growcontinuously in culture).These hybrid cells are selected for growthand secretion of Ig; subsequently, they are further screened andselected for the production of the desired Ig.Monoclonal antibod-ies are used extensively for research and diagnostics, e.g., devel-opment of ELISA kits for detection of human chorionic gonadot-ropin in the urine of pregnant women, detecting immune cellnumbers (e.g., CD4+ T cells in blood) during pathological condi-tions, e.g.,AIDS. In addition, such antibodies are used for therapy,e.g., treatment with an Ig (known as Herceptin) to an oncoprotein(c-erbB-2) is effective against some breast cancers. Also, one ofthe lines of treatment of B-cell lymphomas is using an Ig to CD20known as Rituximab.

Consequences of Reduced or Excess Antibody Produc-tion

As seen in the above section, antibodies play several roles and toevaluate their physiological roles, it is useful to study the effects ofreduced or increased production of antibodies. One such defi-ciencydisease isBruton’sX-linked agammaglobulinemia (XLA),where amale child is unable to produce Ig (Box 3). This defectivegene XLA encodes Bruton’s tyrosine kinase (Btk) which plays arole inBcellmaturation.Due tomutations inBtk, patients possessvery low levels of immunoglobulins in blood and suffer fromrecurrent infections duringchildhood.Another immunodeficiencycondition is ‘Common Variable Immunodeficiency’ (CVID) inwhich reduced amounts of antibodies are found. The symptomsobserved are similar to XLA but the reasons for this are multiple,due tomutations indifferent genes involved inantibodyproduction.

Serum Ig arepolyclonal as theyoriginate fromdifferentplasmacells.

Antibodies playseveral roles and toevaluate theirphysiological roles,it is useful to studythe effects ofreducedorincreasedproductionofantibodies.


GENERAL � ARTICLE

Box 3. Some Disorders due to Mutations or Deficiencies in Key Immune Response Genes

Disorder Description

Autoimmune polyendocrinopathy Results due to mutations in the autoimmune regulator, AIRE.– candidiasis – ectodermal Studies on AIRE in mice have revealed that it isdystrophy highly expressed in thymic medullary epithelial tissue(APECED) where it regulates expression of peripheral tissue

antigens. Individuals suffering from APECED displaychronic mucocutaneous candidiasis, Type I diabetes andother autoimmune features.

DiGeorge Syndrome Characterized by an absence of the thymus and afflictedindividuals are immunodeficient, suffer from hypoparathyroidism,congenital heart disease and display characteristic facial abnormalities.

SevereCombined It is a broad family of disorders characterized by both BImmunodeficiency and T cell deficiencies that may develop due to a variety(SCID) of reasons. The most common cause of SCID is mutation in the

common gamma chain of the cytokine receptors for IL-2, IL-4, IL-7,IL-9, IL-15 and IL-21. The second most common reason isAdenosine deaminase (ADA) deficiency, which affects purinemetabolism and reduces lymphocyte proliferation. Mutations inRAG affecting VDJ recombination also cause SCID. Most SCIDpatients suffer from recurrent infections and usually die early in lifedue to absence of the adaptive immune response.

X-linked hyper Characterized by abnormally high levels of IgM and absence ofIgM syndrome IgA, IgE and IgG. The defect arises due to inability of T-helper cells

to express CD40L. Therefore, T-dependent B cell activation andclass switching to other antibody isotypes does not occur. Affectedindividuals suffer recurrent infections especially those associatedwith the respiratory tract.

X-linkedAgammaglobulinemia Mature B cells fail to develop from pre-B cells due to defects in a(XLA) signal transduction component known as Bruton’s Tyrosine Kinase

(Btk). Characterized by very low levels of IgG and absence of otherisotypes. People suffering from XLA lack peripheral B cells and cannotmake Ig. Consequently, they suffer from recurrent bacterial infections.

Bare Lymphocyte Syndrome Occurs due to low or no surface MHC class I or class II expression.Patients have low numbers of peripheral T lymphocytes andsuffer from recurrent infections, especially diarrhoea and candidiasis.This disorder is usually caused due to defects in a transporter (TAP),which is essential for surface expression of MHC class I, ortranscription factors, e.g. CIITA, required for MHC class II expression.


GENERAL � ARTICLE

In general, plasmacells have a short life-span although some long-lived plasma cells have also been found. Plasma cells have a finitelife span; however, in cases when a plasma cell becomes tumori-genic, i.e., keeps on dividing even without antigen stimulation, itgives rise to a condition known as ‘multiple myeloma’. Hereimmortal plasma cells secrete Ig in great excess which are termedas myeloma proteins and constitute about 95% of serum antibod-ies. In some cases, myeloma cells secrete huge amounts of lightchains which are named ‘Bence-Jones proteins’ (after the nameof the physician who made this observation). Interestingly, se-quencingofBence-Jones proteins played a pivotal role in elucida-tion of the amino acid sequence of immunoglobulin.

The secondpart of the articlewill discuss cell-mediated immunity.

Acknowledgments

We thank Profs. R Manjunath, P Sadhale and S Raghavan forpatient reading and comments on themanuscript. The encourage-ment of allmembers of theDpN laboratory is appreciated.We aregrateful to the Government of India agencies (DBT, CSIR, DSTand ICMR) for providing research funding to the laboratory.

Suggested Reading

[1] F M Burnet, The clonal selection theory of acquired immunity,pp.1–209, Cambridge University Press, Cambridge, 1959.

[2] T S Chang, B Glick, A R Winter, The significance of the bursa ofFabricius of chickens in antibody production., Poultry Sci. Vol.34,p.1187, 1955.

[3] N Hozumi, S Tonegawa, Evidence for somatic rearrangement ofimmunoglobulin genes coding for variable and constant regions.Proc. Natl. Acad. Sci., USA., Vol.73, pp.3628–3632, 1976.

[4] R M Zinkernagel and P C Doherty, Immunological surveillanceagainst altered self components by sensitised T lymphocytes inlymphocytic choriomeningitis, Nature, Vol.251, pp.547–548, 1974.

[5] P Bretscher and M Cohn, A theory of self-nonself discrimination,Science , Vol.169, pp.1042–1049, 1970.

[6] J F Miller, Immunological function of the thymus, Lancet,Vol.2, pp.748–749, 1961.

Address for CorrespondenceDipankarNandi

Department of BiochemistryIndian Institute of ScienceBangalore 560 012, India.

Email:[email protected]

the major players in adaptive immunity

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