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1) Immunological tolerance: i) One of the remarkable properties of the normal immune system is that it can react to an enormous
variety of microbes but does not react against the individual’s own (self) antigens. Ii) This responsesiveness to self antigens, also called
immunological tolerance, is maintained despite the fact that the molecular mechanisms by which lymphocyte receptor specificities
are generated are not biased to exclude receptors for self antigens.
2) Central T lymphocyte tolerance: i) The principal mechanisms of central tolerance in T cells are death of immature T cells and the
generation of CD4+ regulatory T cells. Ii) If a lymphocyte that has not completed its maturation interacts strongly with a self antigen,
displayed as a peptide bound to a self major histocompatibility complex (MHC) molecule, that lymphocyte receives signals that trigger
apoptosis. Thus, the self-reactive cell dies before it can become functionally competent. This process, called negative selection is a
major mechanism of central tolerance. Iii) The process of negative selection affects self-reactive CD4+ T cells and CD8+ T cells, which
recognize self peptides displayed by class II MHC and class I MHC molecules. Iv) many self proteins that are normally present only in
certain peripheral tissues are also expressed in some of the epithelial cells of the thymus. V) A protein called AIRE (autoimmune
regulator) is responsible for the thymic expression of these peripheral tissue antigens. Mutations in the AIRE gene are the cause of a
rare disorder called autoimmune polyendocrine syndrome.
3) Peripheral T Lymphocyte tolerance: i) Peripheral tolerance is induced when mature T cells recognize self antigens in peripheral
tissues, leading to functional inactivation (anergy) or death, or when the self-reactive lymphocytes are suppressed by regulatory T
cells. Ii) Peripheral tolerance is clearly important for preventing T cell responses to self antigens that are not present in the thymus,
and it also may provide backup mechanisms for preventing autoimmunity in situations where central tolerance is incomplete. Iii)
Antigen recognition without adequate costimulation results in T cell anergy or death, or makes T cells sensitive to suppression by
regulatory T cells. Iv) T lymphocytes need at least two signals to induce their proliferation and differentiation into effector and
memory cells: Signal 1 is always antigen, and signal 2 is provided by costimulators that are expressed on Antigen-presenting cells
(APCs). V) Dendritic cells in uninfected tissues and organs are immature and express minimal costimulators like B7 proteins. They
process and display self-antigens, but lack strong costimulation due to an innate immune response. Vi) The presence or absence of

costimulation determines T cell activation or tolerization. 🌑mechanisms of peripheral T cell tolerance: 1) Anergy: Anergy in T cells
refers to long-lived functional unresponsiveness that is induced when these cells recognize self antigens. Ii) Self-antigens display low
costimulators, leading to anergy induction through inadequate recognition, causing anergic cells to survive but not respond. Iii) The
two best-defined mechanisms responsible for the induction of anergy are abnormal signaling by the TCR complex and the delivery of
inhibitory signals from receptors other than the TCR complex. Iv) When T cells recognize antigens without costimulation, the TCR
complex may lose its ability to transmit activating signals. In some cases, this is related to the activation of enzymes (ubiquitin ligases)
that modify signaling proteins and target them for intracellular destruction by proteases. V) On recognition of self antigens, T cells also
may preferentially engage one of the inhibitory receptors of the CD28 family, cytotoxic T lymphocyte–associated antigen 4 (CTLA-4, or
CD152) or programmed death protein 1 (PD-1), Anergic T cells may express higher levels of these inhibitory receptors, which will
inhibit responses to subsequent antigen recognition. ❌ Regulation of T Cell Responses by Inhibitory Receptors: Immune responses is
the balance between activating and inhibiting receptors including NK cells, B- lymphocytes and T-cells. In T cells, the best-defined
inhibitory receptors are CTLA-4 and PD-1. 1) CTLA-4: i) CTLA-4 is a protein that is expressed on both activated CD4+ T cells and
regulatory T cells. It works by blocking and removing B7 molecules from the surface of APCs, reducing costimulation and preventing T
cell activation. CTLA-4 may also deliver inhibitory signals to T cells. It recognizes the same B7 costimulators that bind to CD28 and
initiate T cell activation. One theory explains why T cells choose CD28 or CTLA-4 differently is that CTLA-4 has a higher affinity for B7
molecules than CD28. When B7 levels are low, CTLA-4 is preferred, while when B7 levels are high, CD28 is engaged more. 2) PD-1: PD-
1 is expressed on CD4+ and CD8+ T cells after antigen stimulation. It has an immunoreceptor tyrosine-based inhibitory motif (ITIM)
typical of receptors that deliver inhibitory signals. PD-1 terminates responses of T cells to self antigens and also to chronic infections,
notably virus infections. ❌ Checkpoint blockade therapy for cancer treatment: i) Cancer patients are treated with antibodies that
block the function of inhibitory receptors, enhancing antitumor immune responses and tumor regression. Ii) This therapy, known as
checkpoint blockade, removes the brakes on immune responses, causing autoimmune reactions in some patients. Iii) The treatment is
considered to be effective in keeping autoreactive T cells in check, as inhibitory receptors are constantly in action. 2) Suppression: i)
Regulatory T cells develop in the thymus or peripheral tissues on recognition of self antigens and suppress the activation of potentially
harmful lymphocytes specific for these self antigens. Ii) The majority of self-reactive regulatory T cells probably develop in the thymus,
but they may also arise in peripheral lymphoid organs. Iii) Most regulatory T cells are CD4+ and express high levels of CD25, the α
chain of the interleukin-2 (IL-2) receptor. Iv) They also express a transcription factor called FoxP3, which is required for the
development and function of the cells. Mutations of the gene encoding FoxP3 in humans or in mice cause a systemic, multiorgan
autoimmune disease, demonstrating the importance of FoxP3+ regulatory T cells for the maintenance of self-tolerance. V) The
survival and function of regulatory T cells are dependent on the cytokine IL-2. Vi) The cytokine transforming growth factor β (TGF-β)
also plays a role in the generation of regulatory T cells, perhaps by stimulating expression of the FoxP3 transcription factor.
Signals for T-cell to proliferation: i) T lymphocytes need at least two signals to induce their proliferation and differentiation into
effector and memory cells: a) Signal 1 is always antigen b) signal 2 is provided by costimulators that are expressed on antigen-
presenting cells (APCs), typically as part of the innate immune response to microbes dendritic cells in normal uninfected tissues and
peripheral lymphoid organs are in a resting (or immature) state, in which they express little or no costimulators, such as B7 proteins.
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Ii) These dendritic cells may constantly process and display the self antigens that are present in the tissues. Iii) lymphocytes with
receptors for the self antigens are able to recognize the antigens and thus receive signals from their antigen receptors (signal 1). Iv)
the T cells do not receive strong costimulation because there is no accompanying innate immune response. V) The presence or
absence of costimulation is a major factor determining whether T cells are activated or tolerized.
4) Antigen presenting cells reinforce self tolerance/Abundant self antigens induce peripheral deletion: 1) autoantigen at low
concentration: Immunological ignorance predominantly works if the self antigen is present at relatively low concentrations. The
antigen remains in the tissue in which it is expressed and is not transported into the draining lymph node under steady state
conditions. 2) autoantigen at high concentration: If tissue antigens are present at high concentrations they will be brought to draining
lymph nodes by DC even under steady state (non-inflammatory) conditions. Under these conditions, the DC are not immunogenic but
tolerogenic and induce apoptosis or anergy in T cells that recognize the self Ag.
5) Natural Treg cells differentiate in the thymus / Natural and induced T-regulatory cells: i) In mice CD4- CD8- double negative (DN) T
cell precursors enter the thymus and undergo several selection processes. Ii) At the CD4 single positive stage a population of
thymocytes starts expressing FoxP3 and CD25. Iii) These cells leave the thymus as natural regulatoryCD4 single positive stage a
population of thymocytes starts expressing FoxP3 and CD25. Iv) These cells leave the thymus as natural regulatory T cells (Treg) which
comprise peripheral CD4+ T cells. The CD4+ FoxP3- CD25- thymocytes emigrate from the thymus as naïve effector TH cells. V) Upon
activation and instruction by APC they can assume different effector functions, broadly characterized as TH1, TH2, or TH17 cells. Vi)
Some of the naïve CD4+ FoxP3- CD25- TH cells can also develop into CD4+ FoxP3+ CD25+ induced Treg cells. These iTregs exert similar
functions to the nTregs, however their FoxP3 expression is less stable.
6) iTreg cells differentiate in the periphery: i) Induced Treg cells (iTregs) are mature T cells that can acquire Treg phenotype and
function outside the thymus. Ii) FoxP3 expression can be induced in naïve CD4þ cells in vitro by antigen recognition in the presence of
TGF-b. iii) There is a close developmental relationship between iTregs and TH17 cells. Antigen recognition in the presence of TGF-b
induces FoxP3 expression if IL-6 is not present. Iv) Conversely, antigen recognition in the presence of TGF-b and IL-6 prevents FoxP3
expression and induces RORgt expression, leading to TH17 differentiation. V) The transcription factor IRF4 is necessary for down-
regulating TGF-b-induced FoxP3 expression in response to IL-6. Vi) Chronic antigen stimulation in vivo also induces FoxP3 expression
and iTreg differentiation. Vii) However, iTreg differentiation is less stable and may lose regulatory capacities. In contrast, induction of
FoxP3 by TGF-b does not induce regulatory capacities in human T cells.
7) Treg effector functions: i) Once activated, Treg cells suppress immune responses independent of their own antigen-specificity. This
antigen-nonspecific immunosuppression has been called bystander suppression. Ii) Another characteristic of Treg action has long been
known as infectious tolerance. Iii) The concept of infectious tolerance is based on in vivo transfer studies in which the adoptive
transfer of Tregs induced the differentiation or selective outgrowth of Tregs in the host.
8) Treg effector mechanisms/ Tregs secrete immunosuppressive cytokines: i) Treg cells employ different mechanisms to control
effector T cells (T*). Ii) These include the secretion of immunosuppressive cytokines; the consumption of IL-2, leading to a lack of IL-2
to sustain proliferation and survival of effector T cells leading to Bim-mediated apoptosis, cytolysis of effector T cells and the
modulation of DC maturation and function. IDO, indoleamine 2,3-dioxygenase.
9) Regulatory T cells may suppress immune responses by several mechanisms: i) Some regulatory cells produce cytokines (e.g., IL-10,
TGF-β) that inhibit the activation of lymphocytes, dendritic cells, and macrophages. Ii) Regulatory cells express CTLA-4, which, as
discussed earlier, may block or remove B7 molecules made by APCs and make these APCs incapable of providing costimulation via
CD28 and activating T cells. Iii) Regulatory T cells, by virtue of the high level of expression of the IL-2 receptor, may bind and consume
this essential T cell growth factor, thus reducing its availability for responding T cells. Iv) The great interest in regulatory T cells has in
part been driven by the hypothesis that the underlying abnormality in some autoimmune diseases in humans is defective regulatory T
cell function or the resistance of pathogenic T cells to regulation. V) The importance of defects in regulatory T cells in common human
autoimmune diseases is not established, perhaps because it has proved difficult to identify regulatory T cells specific for self antigens
in humans. There is also growing interest in cellular therapy with regulatory T cells to treat graftversus-host disease, graft rejection,
and autoimmune disorders.
10) Development and function of regulatory T cells: i) CD4+ T cells that recognize self antigens may differentiate into regulatory cells
in the thymus or peripheral tissues, in a process that is dependent on the transcription factor FoxP3. Ii) The larger arrow from the
thymus, compared to the one from peripheral tissues, indicates that most of these cells probably arise in the thymus. Iii) These
regulatory cells inhibit the activation of naïve T cells and their differentiation into effector T cells by contact-dependent mechanisms
or by secreting cytokines that inhibit T cell responses. Iv) The generation and maintenance of regulatory T cells also require

interleukin-2 (not shown). DC, Dendritic cell. 🌑T cell Anergy: i) T cells that cannot be completely activated upon recognition of their
cognate antigen are called anergic. Ii) The phenomenon of T cell clonal anergy was discovered in CD4+ TH1 clones that failed to
produce IL-2 and proliferate when stimulated in vitro with antigen in the absence of costimulatory signals. Iii) TNF-family members
such as OX40 and 4-1BB and several cytokines, including the gamma-chain cytokines IL-2, -4, -7 and -15 can contribute to resistance of
effector T cells against suppression by Tregs.
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🌑Deletion: Apoptosis of Mature Lymphocytes: i) Recognition of self antigens may trigger pathways of apoptosis that result in
elimination (deletion) of the self-reactive lymphocytes. Ii) There are two likely mechanisms of death of mature T lymphocytes induced
by self antigens: a) Antigen recognition triggers the production of pro-apoptotic proteins in T cells, leading to cell death. In normal
immune responses, these proteins are counteracted by anti-apoptotic proteins induced by costimulation and growth factors. Self-
administered antigens do not stimulate anti-apoptotic protein production, and a lack of survival signals causes cell death in those that
recognize these antigens. B) Self-tolerance involves the coexpression of death receptors and their ligands, which trigger caspases and
apoptosis. The Fas protein (CD95) and Fas ligand (FasL) are involved in self-tolerance, expressed on various cell types and mainly on
activated T cells. The Fas pathway may also be involved in the death of some B cells in germinal centers.
11) Extrinsic Pathway: i) Fas ligand on the surface of a killer lymphocyte activates Fas death receptors on the surface of the target cell.
Both the ligand and receptor are homotrimers. Ii) The cytosolic tail of Fas then recruits the adaptor protein FADD via the death
domain on each protein (FADD stands for Fas-associated death domain). Iii) Each FADD protein then recruits an initiator procaspase
(procaspase-8, procaspase-10, or both) via a death effector domain on both FADD and the procaspase, forming a death-inducing
signaling complex (DISC). Iv) Within the DISC, the initiator procaspase molecules are brought into close proximity, which activates
them; the activated procaspases then cleave one another to stabilize the activated protease, which is now a caspase. V) Activated
caspase-8 and caspase-10 then cleave and activate executioner procaspases, producing a caspase cascade, which leads to apoptosis.
12) Intrinsic Pathway: i) The binding of cytochrome c causes the Apaf 1 to hydrolyze its bound dATP to dADP. The replacement of the
dADP with dATP or ATP (not shown) then induces the complex of Apaf 1 and cytochrome c to aggregate to form a large. Ii) Heptameric
apoptosome, which then recruits procaspase-9 through a caspase recruitment domain (CARD) in each protein. Iii) The procaspase-9
molecules are activated within the apoptosome and iv) are now able to cleave and activate downstream executioner procaspases,
which leads to the cleavage and activation of these molecules in a caspase cascade. V) Other proteins released from the mitochondrial
intermembrane space.
13) Mechanisms of apoptosis of T lymphocytes: i) T cells respond to antigen presented by normal antigen presenting cells (APCs) by
secreting interleukin-2 (IL-2), expressing anti-apoptotic (pro-survival) proteins, and undergoing proliferation and differentiation. Ii) The
anti-apoptotic proteins prevent the release of mediators of apoptosis from mitochondria. Iii) Self antigen recognition by T cells
without costimulation may lead to relative deficiency of intracellular anti-apoptotic proteins, iv) the excess of pro-apoptotic proteins
causes cell death by inducing release of mediators of apoptosis from mitochondria (death by the mitochondrial [intrinsic] pathway of
apoptosis). V) Alternatively, self antigen recognition may lead to expression of death receptors and their ligands, such as Fas and Fas
ligand (FasL), on lymphocytes, and engagement of the death receptor leads to apoptosis of the cells by the death receptor (extrinsic)
pathway.
14) Self antigens differ from foreign microbial antigens: i) Self antigens are present in the thymus, where they induce deletion and
generate regulatory T cells; by contrast, most microbial antigens tend to be excluded from the thymus, because they are typically
captured from their sites of entry and transported into peripheral lymphoid organs. Ii) Self antigens are displayed by resting APCs in
the absence of innate immunity and second signals, thus favoring the induction of T cell anergy or death, or suppression by regulatory
T cells. By contrast, microbes elicit innate immune reactions, leading to the expression of costimulators and cytokines that promote T
cell proliferation and differentiation into effector cells. Iii) Self antigens are present throughout life and may therefore cause
prolonged or repeated TCR engagement, again promoting anergy, apoptosis, and the development of regulatory T cells.
15) B Lymphocyte Tolerance: i) Self polysaccharides, lipids, and nucleic acids are T-independent antigens that are not recognized by T
cells. These antigens must induce tolerance in B lymphocytes to prevent autoantibody production. Ii) Self proteins may not elicit
autoantibody responses because of tolerance in helper T cells and in B cells. Iii) It is suspected that diseases associated with
autoantibody production, such as systemic lupus erythematosus, are caused by defective tolerance in both B lymphocytes and helper
T cells.
16) Central B Cell Tolerance: i) When immature B lymphocytes interact strongly with self antigens in the bone marrow, the B cells
either change their receptor specificity (receptor editing) or are killed (deletion). Ii) An immature B cell that recognizes self antigen in
the bone marrow changes its antigen receptor (receptor editing), dies by apoptosis (negative selection, or deletion), or reduces
antigen receptor expression and becomes functionally unresponsive. A) Receptor editing: i) Some immature B cells that recognize self
antigens in the bone marrow may reexpress RAG genes, resume immunoglobulin (Ig) light-chain gene recombination, and express a
new Ig light chain. Ii) This new light chain associates with the previously expressed Ig heavy chain to produce a new antigen receptor
that may no longer be specific for the self antigen. Iii) This process of changing receptor specificity, called receptor editing. B)
Deletion: i) If editing fails, immature B cells that strongly recognize self antigens receive death signals and die by apoptosis. This
process of deletion is similar to negative selection of immature T lymphocytes. Ii) As in the T cell compartment, negative selection of B
cells eliminates lymphocytes with high-affinity receptors for abundant, and usually widely expressed, cell membrane or soluble self
antigens. C) Anergy: Some self antigens, such as soluble proteins, may be recognized in the bone marrow with low avidity. B cells
specific for these antigens survive, but antigen receptor expression is reduced, and the cells become functionally unresponsive
(anergic).
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17) Peripheral B Cell Tolerance: i) Mature B lymphocytes that encounter self antigens in peripheral lymphoid tissues become
incapable of responding to that antigen. Ii) one hypothesis, if B cells recognize an antigen and do not receive T cell help (because
helper T cells have been eliminated or are tolerant), the B cells become anergic because of a block in signaling from the antigen
receptor. Anergic B cells may leave lymphoid follicles and are subsequently excluded from the follicles. Iii) These excluded B cells may
die because they do not receive necessary survival stimuli. Iv) B cells that recognize self antigens in the periphery may also undergo
apoptosis, or inhibitory receptors on the B cells may be engaged, thus preventing activation, regulatory T cells may also contribute to
B cell tolerance.
18) Tolerance to commensal microbes and fetal antigens: i) The evolution of placentation in eutherian mammals has led to the
maturation of the fetus before birth, but it also creates a problem where foreign paternal antigens must be tolerated by the mother’s
immune system. Ii) One mechanism of this tolerance is the generation of peripheral FoxP3+ regulatory T cells specific for these
antigens. Iii) Placentation is strongly correlated with the ability to generate stable peripheral regulatory T cells. Iv) It is unclear
whether women who suffer recurrent pregnancy losses have a defect in the generation or maintenance of these regulatory T cells.
V) Other mechanisms of fetal tolerance include the exclusion of inflammatory cells from the pregnant uterus, poor antigen
presentation in the placenta, and an inability to generate harmful Th1 responses in the healthy pregnant uterus.
19) Complement Components: I) The complement system consists of proteins and glycoproteins, primarily produced by liver
hepatocytes, blood monocytes, tissue macrophages, and epithelial cells of the gastrointestinal and gastrointestinal tracts. Ii) These
components make up 5% of the serum globulin fraction and circulate in functionally inactive forms as proenzymes or zymogens. Iii)
The complement-reaction sequence starts with an enzyme cascade. Iv) Complement components are designated by numerals, letter
symbols, or trivial names. V) Peptide fragments formed by activation are denoted by small letters. Vi) The larger fragments bind to the
target near the activation site, while smaller fragments diffuse from the site and can initiate localized inflammatory responses by
binding to specific receptors. Vii) Complement fragments interact to form functional complexes, with complexes with enzymatic
activity designated by a bar over the number or symbol. Function of Complement: i) Research on complement now includes more
than 30 soluble and cell-bound proteins. ii) The biological activities of this system affect both innate and acquired immunity and reach
far beyond the original observations of antibody- mediated lysis of bacteria and red blood cells. iii) Lysis of cells, bacteria, and viruses
Opsonization, which promotes phagocytosis of particulate antigens. Iv) Binding to specific complement receptors on cells of the
immune system, triggering specific cell functions, inflammation, and secretion of immune regulatory molecules. v) Immune clearance,
which removes immune complexes from the circulation and deposits them in the spleen and liver.
20) The Complement Pathway: 1) The Classical Pathway Antigen-Antibody dependent / mediated pathway: i) C1q binding to Fc
binding sites converts C1r to an active serine protease enzyme, C1r, which cleaves C1s to a similar enzyme, C1s. ii) C1s has two
substrates, C4 and C2. C4 is an aglycoprotein that is activated when C1 hydrolyzes it into small fragments (C4a) and larger fragments
(C4b). iii) The C2 proenzyme attaches to the exposed binding site on C4b, where it is cleaved by C1 and the smaller fragment (C2b)
diffuses away. Iv) The resulting C4b2a complex is called C3 convertase, which converts C3 into an active form. The smaller fragment,
C4a, is an anaphylatoxin. V) Hydrolysis of C3 component by C3 convertase generates short fragments (C3a) and C3b, resulting in
significant amplification. vi) Some C3b binds to C4b2aC3b to form a trimolecular complex called C5 convertase. Vii) The C3b
component of this complex binds C5 and alters its conformation, allowing C5 to be cleaved into C5a and C5b, initiating the formation
of the membrane attack complex.
21) Alternative Pathway / Antibody-Independent: i) In the alternative pathway, serum C3, with an unstable thioester bond,
undergoes slow spontaneous hydrolysis to produce C3a and C3b. Foreign cell C3b binds factor B, which cleaves C3b-bound factor B,
releasing a fragment (Ba) and generating C3bBb. Ii) This C3bBb complex has C3 convertase activity, similar to the classical C4b2a
complex. The C3bBb complex can activate unhydrolyzed C3 to generate more C3b autocatalytically, repeating and amplifying the
initial steps. Iii) The C3bBb3b complex exhibits C5 convertase activity, similar to the classical C4b2a3b complex. Iv) The nonenzymatic
C3b component binds C5, and the Bb component hydrolyzes the bound C5 to generate C5a and C5b, which binds to the antigenic
surface.
22) Lectin Pathway: i) Lectins are proteins that recognize and bind to specific carbohydrate targets, known as the MBL lectin pathway
or mannan-binding lectin pathway. Ii) This pathway, similar to the alternative pathway, does not require antibody activation but
follows a classical mechanism, involving C4 and C2 to produce a C5 convertase. Iii) Mannose-binding lectin (MBL) is activated by
binding to mannose residues on glycoproteins or carbohydrates on microorganisms, including Salmonella, Listeria, Neisseria,
Cryptococcus neoformans, and Candida albicans. Iv) MBL-associated serine proteases, MASP-1 and MASP-2, bind to MBL, causing the
active complex to cleavage and activation of C4 and C2. V) This innate defense mechanism is comparable to the alternative pathway
but utilizes elements of the classical pathway, except for the C1 proteins.
23) Immunomodulation: i) Immunomodulation refers to the use of biological response modifiers (BRMs) to treat cancer by improving
host defense mechanisms against tumors. Ii) These BRMs have a direct antiproliferative effect on tumor cells and enhance the host’s
tolerance to toxic chemicals used to destroy the cancer. Iii) They could provide an alternative to conventional chemotherapy for
various diseased conditions, especially when immune responsiveness is impaired or selective immunosuppression is needed in cases
like infectious diseases, autoimmune disorders, and organ/bone marrow transplantation. Iv) All three classes of immunomodulators—
biologicals, chemical, and cytokines—will continue to play a significant role in advancing and improving the quality of treatment for
various human and animal diseases. Biological response modifiers (BRM): i) intrinsic products that alter immune defenses to
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enhance, direct or restore the body’s ability to fight disease. The patients are given substances called biological response modifiers
such as ã-interferon which activate their immune system, slow the growth of tumors and help in destroying the tumor. Ii) Biological
response modifiers used in biological therapy include: Interleukins, Monoclonal antibodies, Erythropoietin, Tumor necrosis factor.
24) Regulation of the Complement system: A) Before assembly of convertase activity: I) The glycoprotein C1 inhibitor (C1Inh) can
form a complex with C1r2s2, preventing further activation of C4 or C2. Ii) The association of C4b and C2a is blocked by binding C4b-
binding protein (C4bBP), complement receptor type I, or membrane cofactor protein (MCP). Iii) These regulatory proteins, including
soluble C4b-binding protein (C4bBP) and two membrane-bound proteins, complement receptor type 1 (CR1) and membrane cofactor
protein (MCP), bind to C4b and prevent its association with C2a. Iv) Once bound, factor I cleaves C4b into bound C4d and soluble C4c.
V) A similar regulatory sequence prevents assembly of the C3 convertase C3bBb in the alternative pathway. Vi) CR1, MCP, or factor H
bind to C3b and prevent its association with factor B. Vii) Factor I cleaves C3b into a bound iC3b fragment and a soluble C3f fragment.
B) After assembly of convertase: i) Several regulator of complement activation proteins, including C4bBP, CR1, and factor H, act on
the assembled C3 convertase, causing it to dissociate. Ii) DAF, a glycoprotein covalently linked to a glycophospholipid membrane
protein, can also dissociate C3 convertase. Iii) These proteins accelerate the decay of C3 convertase by releasing the component with
enzymatic activity (C2a or Bb) from the cell-bound component (C4b or C3b). Iv) Once dissociation occurs, factor I cleaves the
remaining membrane-bound C4b or C3b component, irreversibly inactivating the convertase. C) Regulation at assembly of
membrane-attack complex (MAC): i) Regulatory proteins play a crucial role in the membrane-attack complex, preventing innocent-
bystander lysis to healthy cells. Ii) Serum proteins, such as S protein, bind to released C5b67 and prevent its insertion into nearby cells'
membranes. Iii) Complement-mediated lysis is more effective when the complement is from a different species than the cells being
lysed. Iv) Two membrane proteins, homologous restriction factor (HRF) and membrane inhibitor of reactive lysis (MIRL or CD59), block
MAC formation, protecting cells from nonspecific complement-mediated lysis by binding to C8, preventing assembly of poly-C9 and its
inclusion into the plasma membrane.
25) Regulation of Classical pathway / antigen antibodies dependent: i) the glycoprotein C1 inhibitor (C1Inh) can form a complex with
C1r2s2, causing it to dissociate from C1q and preventing further activation of C4 or C2. Ii) Association of C4b and C2a is blocked by
binding C4b-binding protein (C4bBP), complement receptor type I, or membrane cofactor protein (MCP). Iii) These regulatory proteins
include soluble C4b-binding protein (C4bBP) and two membrane-bound proteins, complement receptor type 1 (CR1) and membrane
cofactor protein (MCP). Iv) Each of these regulatory proteins binds to C4b and prevents its association with C2a. V) Once C4bBP, CR1,
or MCP is bound to C4b, another regulatory protein, factor I, cleaves the C4b into bound C4d and soluble C4c.
26) Regulation of alternative Pathway / antibody independent: i) A similar regulatory sequence operates to prevent assembly of the
C3 convertase C3bBb in the alternative pathway. Ii) In this case CR1, MCP, or a regulatory component called factor H binds to C3b and
prevents its association with factor B. Iii) Once CR1, MCP, or factor H is bound to C3b, factor I cleaves the C3b into a bound iC3b
fragment and a soluble C3f fragment. Iv) Further cleavage of iC3b by factor I releases C3c and leaves C3dg bound to the membrane..
27) Cytokine Secretion by TH1 and TH2 Subsets: i) The immune response to a pathogen involves a range of effector functions to
eliminate the disease agent or its toxic products from the host. Ii) Different cytokine-secretion patterns among TH-cell subsets
determine the type of immune response to a specific antigenic challenge. Iii) CD4+ TH cells primarily helper functions through
secreted cytokines, which either act on the cells producing them or modulate responses through paracrine pathways. Iv) CD8+ CTLs
also secrete cytokines, but their array is more restricted than CD4+ TH cells. V) Two CD4+ TH-cell subpopulations, TH1 and TH2, can be
distinguished by their cytokine. Vi) TH1 and TH2 cells are characterized by the following functional differences: a) The TH1 subset is
responsible for many cell-mediated functions (e.g., delayed-type hypersensitivity and activation of TC cells) and for the production of
opsonization promoting IgG antibodies (i.e. antibodies that bind to the high-affinity Fc receptors of phagocytes and interact with the
complement system). This subset is also associated with the promotion of excessive inflammation and tissue injury. b) The TH2 subset
stimulates eosinophil activation and differentiation, provides help to B cells, and promotes the production of relatively large amounts
of IgM, IgE, and non complement activating IgG isotypes. The TH2 subset also supports allergic reactions.
28) cytokine of the TH1 subset: 1) IFN-γ - i) activates macrophages, stimulating these cells to increase microbicidal activity, up-
regulate the level of class II MHC. Ii) TH1 cells secrete IFN-γ, which triggers antibody-class switching to IgG classes, such as IgG2a in
mice, which aid in phagocytosis and complement fixation. 2) IL-12 - which induces TH cells to differentiate into the TH1 subset. 3) TNF
& IFN-γ - cytokines that mediate inflammation, and it is their secretion that accounts for the association of TH1 cells with
inflammatory phenomena such as delayed hypersensitivity. 4) IL-2 and IFN-γ - promote the differentiation of fully cytotoxic TC cells
from CD8+ precursors.
29) cytokine of the TH2 subset: 1) IL-4 and IL-5 by cells of the TH2 subset induces production of IgE and supports eosinophil-mediated
attack on helminth (roundworm) infections. 2) IL-4 - class switching that produces IgG increases the extent to which B cells switch
from IgM to IgE. 3) IL-4 and IL-10 - suppress the expansion of TH1 cell populations. TH1 and TH2 cells is independently. Many helper T
cells do not show either a TH1 or a TH2 any helper T cells do not show either a TH1 or a TH2 profile, One of the best described of
these is the TH0 subset, which secretes IL-2, IL-4, IL-5, IFN-, and IL-10, as well as IL-3 and GM-CSF.
30) The Development of TH1 and TH2 Subsets Is Determined by the Cytokine Environment: Cytokine-mediated generation and cross
regulation of TH subsets. Antigen-activated naive CD4+ T cell produces IL-2 and proliferates. If it proliferates in an IL-12 dominated
environment, it generates a population of TH1 cells that secretes a characteristic profile of cytokines including interferon Y. A positive
feedback loop is established when IFN-Y secreted by the expanding TH1 population stimulates dendritic cells or macrophages to
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produce more IL-12. If the environment is dominated by IL-4, a TH2 population emerges and secretes a profile of cytokines that
promotes eosinophil activation and the synthesis of certain antibody classes. Key cytokines produced by each subset positively
regulate the subset that produces it and negatively regulate the other subset.
31) Cytokine Profiles Are Cross-Regulated: Critical cytokines produced by TH1 and TH2 subsets have two effects on subset
development: promoting the growth of the subset that produces them and inhibiting the development and activity of the opposite
subset, known as cross-regulation. IFN-` (secreted by the TH1 subset) preferentially inhibits the proliferation of the TH2 subset, while
IL-4 and IL-10 down-regulate the secretion of IL-12, a critical cytokine for TH1 differentiation. These cytokines also have opposing
effects on target cells other than TH subsets. The phenomenon of cross-regulation explains the inverse relationship between antibody
production and cell-mediated immunity. Two transcription factors, T-Bet and GATA-3, are key elements in determining subset
commitment and cross-regulation. The expression of T-Bet drives cells to differentiate into TH1 cells and suppresses their
differentiation along the TH2 pathway. The up-regulation of T-Bet represses the expression of GATA-3, thereby repressing the other.
32) Regulatory T cells: They are CD4+CD25+ Thymus derived T regulatory cells are called natural Treg cells Supress Immune system
against self and non- self antigens. Do not produce effector molecules like IL-2, TNF-alpha, TFN-gamma or IL-4. Supress the
proliferation of effector T cells CD25 also expressed by activated T cells. The transcription factor forkhead box P3 (FoxP3) controls
Treg development.
33) Immunogenicity Vs Antigenicity: I) Immunogenicity: ability to induce humoral and cell mediated immune response. All

Immunogens are antigen. 🌑 B cell + Antigen Effector B Cell +Memory B cell 🌑 T cell+ Antigen Effector T Cell + Memory T cell ii) Ability
to combine specifically the product of humoral or cell mediated immune response. All antigen are not immunogen. Iii) Antigenecity is
the ability to combine specifically with secreted antibodies and surface receptor of T cell. Factors That Influence Immunogenicity: i)
To protect against infectious disease, the immune system must be able to recognize bacteria, bacterial products, fungi, parasites, and
viruses as immunogens. Ii) the immune system actually recognizes particular macromolecules of an infectious agent, generally either
proteins or polysaccharides. Iii) Degree of Immunogenicity Proteins > polysaccharides > lipids and nucleic acid (most immunogenic)
(least immunogenic). Iv) For cell-mediated immunity, only proteins and some lipids and glycolipids serve as immunogens. V) These
molecules are not recognized directly. Proteins must first be processed into small peptides and then presented together with MHC
molecules on the membrane of a cell before they can be recognized as immunogens.

1) Cell culture: Cell culture refers to the removal of cells from an animal or plant and their subsequent growth in a favorable
artificial environment. The cells may be removed from the tissue directly and disaggregated by enzymatic or mechanical means
before cultivation, or they may be derived from a cell line or cell strain that has already been already established.

🌑Types of cell culture: 1) primary cell culture: i) Primary cell culture is the first culture obtained directly from animal tissue through
mechanical, chemical, or enzymatic methods. Ii) These slow-growing cells carry all the characteristics of the original tissue or cells and
have the same number of chromosomes as the original cells. Iii) They are used to preserve and maintain cell growth on artificial
growth mediums at specific conditions. Primary cell cultures can be subcultured to obtain other cultures that continue to grow
indefinitely or die after a few subcultures. Subcultures can introduce mutations, potentially resulting in cell lines. Iv) Primary cell
cultures are difficult to obtain, have a shorter lifespan, and are prone to contamination by bacteria and viruses. V) Primary cell
cultures can be further divided into two groups depending on the kind of cells present in the culture; a) Adherent cells: i) Cells in
culture require a stable, inert surface for adherence and growth, typically solid and nontoxic. Ii) These cells are obtained from organ
tissues, like kidney cells and mouse fibroblast STO cells, which remain immobilized within connective tissue. B) Suspension cells: i)
Cells can grow efficiently as suspensions, requiring no solid surface attachment. Ii) They can be grown on liquid media for fresh
subcultures. Iii) The ability to grow as suspension depends on the source of cells, such as vascular blood cells suspended in plasma.
2) Secondary cell culture: i) Secondary cell cultures are obtained after primary cell cultures are subcultured in fresh media, resulting in
long-lasting cells with regular nutrient availability. Ii) They are preferred over primary cell cultures due to their ease of growth and
preservation. Iii) They are formed through enzymatic treatment, washing, and resuspension in fresh media. Iv) Secondary cultures
maintain optimal cell density for growth. However, they may introduce mutations and genetic alterations, leading to immortal cells. V)
Continuous subculture can reduce the risk of contamination by bacteria and viruses. Vi) Additionally, cells may differentiate over time,
resulting in aberrant cells.
3) Cell line: i) A cell line is a subculture of a primary culture, consisting of a pure cell culture. Ii) It typically exhibits functional
similarities to primary cells, but can be modified in genotype and phenotype. Iii) A cell line can consist of multiple lineages with similar
or different phenotypes. Iv) Cell lines can be further divided into two groups based on the growth patterns of the cells; a) Finite cell
lines: i) Finite cell lines are cultures where cells can divide 20 to 100 times before dying. Ii) The number of divisions and lifespan
depend on factors like lineage differences, species, culture conditions, and media. Iii) These cells grow as adherent cells on solid
surfaces, and their lifespan depends on various factors. B) Continuous cell lines: i) Continuous cell lines are cells that exhibit indefinite
growth through subcultures, forming independent cultures and being immortal. Ii) They can be transformed through genetic
alterations and can be tumorigenic. Iii) These cells can be formed from normal primary cell cultures after treatment with chemical
 7
carcinogens or oncogenic viruses. Iv) They can grow to higher density, form suspensions on liquid media, and even form multilayered
structures on culture vessels. V) These cells can be transformed through genetic alterations or on top of each other.


🌑 Application of cell culture: i) Model System: Cell culture are used as model system to study basic cell biology and biochemistry, to
study the interaction between cell and disease causing agents like bacteria, virus. Ii) Cancer Research- The basic difference between
normal cell and cancer cell can be studied using animal cell culture technique, as both cells can be cultured in laboratory. Iii) Virology:-
Animal cell cultures are used to replicate the viruses instead of animals for the production of vaccine. Cell culture can also be used to
detect and isolate viruses. Iv) Toxicity Testing:- Animal cell culture is used to study the effects of new drugs, cosmetics and chemicals
on survival and growth of a number of types of cells. Especially liver and kidney cells. V) Vaccine Production:-Cultured animal cells are
used in the production of viruses and these viruses are used to produce vaccines. For example vaccines for deadly diseases like polio,
rabies, chicken pox etc. Vi) Genetically Engineered Protein:- Animal cell cultures are used to produce commercially important
genetically engineered proteins such as monoclonal antibodies, insulin, hormones, and much more.

🌑 Advantage: i) the consistency and reproducibility of results that can be obtained from using a batch of clonal cells. Ii) Cell cultures
have a highly control of the physicochemical environment (i.e., pH, temperature, osmotic pressure, oxygen, and carbon dioxide
tension) which can be controlled very accurately, and the control of physiological conditions, which can be constantly examined.

🌑Disadvantages: i) highly skilled personnel, techniques must be performed using strict asepsis techniques because animal cells grow
slower than many of the common contaminants (e.g., bacteria, viruses and fungi). Ii) Additionally, animal cells may not survive when
isolated and therefore are not capable of an independent sustainable existence without providing a complex environment. Iii) One of
the main limitations of cell culture is the expense and effort that has to be applied to obtain a relatively low amount of cells.

Nomenclature of Cell Lines: i) It is a common practice to give codes or designations to cell lines for their identification. For instance,
the code NHB 2-1 represents the cell line from normal human brain, followed by cell strain (or cell line number) 2 and clone number 1.
Ii) While naming the cell lines, it is absolutely necessary to ensure that each cell line designation is unique so that there occurs no
confusion when reports are given in literature. Iii) Further, at the time of publication, the-cell line should be prefixed with a code
designating the laboratory from which it was obtained e.g. NCI for National Cancer Institute, WI for Wistar Institute.

Selecting the Appropriate Cell Line: Consider the following criteria for selecting the appropriate cell line for your experiments: 1)
Species: In general, non-human cell lines have less risk of biohazards, hence preferred. However, species differences need to be taken
into account while extrapolating the data to humans. 2) Functional characteristics: What is the purpose of your experiments? For
example, liver- and kidney-derived cell lines may be more suitable for toxicity testing. 3) Finite or continuous cell lines: Cultures with
continuous cell lines are preferred as they grow faster, easy to clone and maintain, and produce higher yield. But it is doubtful
whether the continuous cell lines express the right and appropriate functions of the cells. Therefore, some workers suggest the use of
finite cell lines, although it is difficult.

Tissue culture media: 1) Eagle’s basal medium (BME) has salt solution, non-essential amino acids, and sodium pyruvate. It is
formulated with a reduced sodium bicarbonate concentration (1,500 mg/l) for use with 5% CO. 2) Dulbecco’s Modified Eagle’s
Medium (DMEM) has twice the concentration of amino acids and four times the amount of vitamins as EMEM. The original
formulation contained 1,000 mg/L of glucose, but in the more commonly used variations this amount was increased to 4,500 mg/L. 3)
Iscove’s Modified Dulbecco’s Medium (IMDM) was formulated for growth of lymphocytes and hybridomas. Compared to DMEM, it
has additional amino acids, vitamins and inorganic salts. Potassium nitrate was substituted for ferric nitrate. It also contains HEPES
and selenium, a reduced sodium bicarbonate concentration (1,500 mg/L) for use with 5% CO. 4) McCoy’s 5A and RPMI-1640 was
originally used to grow Novikoff hepatoma cells and will support the growth of primary cultures. 5) RPMI-1640 is a modification of
McCoy’s 5A and was developed for the long-term culture of peripheral blood lymphocytes. 6) Leibovitz’s L-15 is formulated for use
without CO, incubation. The standard sodium bicarbonate/CO, buffering system is replaced by a combination of phosphate buffers,
free-base amino acids, higher levels of sodium pyruvate, and galactose.

Properties of media: 1) pH: Optimum pH between 7.2 to 7.4 is generally needed for mammalian cells. Phenol red is used as an internal
indicator. 2) Oxygen: Cells depend upon glycolysis for the supply of O2 Selenium controls O2 diffusion. Glutathione acts as free radical
scavenger. 3) Temperature: The optimum temperature of mammal is 37 deg°C Change f plus/minus 5 deg°C is acceptable. 4)
Humidity: For cell growth 100% humidity is essential to reduce evaporation of the media. 5) Antibiotics: penicillin (100U / m/l) for
bacteria. Streptomycin (100mg / m/l) for bacteria, or gentamicin (50mg/ml) for bacteria and nystatin (50mg / m/l) for fungi and yeast
 8
In-vivo systems : experimental animals in Immunology
1) Inbred Strains: i) Inbred strains of mice are produced through 20 or more generations of brother-sister mating, replacing
heterozygosity with homozygosity at all loci. Ii) Repeated inbreeding for 20 generations yields an inbred strain with homozygous
and identical progeny at over 99% of all loci. Iii) These strains have been used to control experimental variation caused by
differences in genetic backgrounds in animals like rats, guinea pigs, hamsters, rabbits, and domestic fowl. Iv) Recombinant inbred
strains of mice involve two inbred strains mated, resulting in a recombination within an interesting locus, such as the MHC locus.
V) Subsequent inbreeding results in an inbred strain of mice bearing part of its MHC from strain A and the other from MHC B. Vi)
This allows animals to determine which subregions contribute to an animal’s immune system properties.

2) Transgenic Animals: i) Transgenic mice are created by introducing cloned foreign genes (transgenes) into mouse embryos. Ii)
These mice allow immunologists to study the expression patterns and functions of numerous transgenes within living animals. Iii)
Researchers can control the transgene’s expression by constructing a transgene with a specific promoter. Iv) For example, a
transgene linked to a metallothionein promoter will only express when zinc is added to their water supply. V) Other promoters
are only functional in certain tissues, like pancreatic cells. Vi) If a transgene is integrated into the chromosomal DNA within a one-
cell mouse embryo, it will be integrated into both somatic and germ-line cells, allowing the transgene to be transmitted to
offspring as a Mendelian trait.

3) Knock-in and Knockout Technologies: i) Knock in and knockout refer to two methods of creating transgenic organisms by either
inserting or completely deactivating genes. Ii) Transgenic mice have a limitation as the transgene is integrated randomly within
the genome, causing some genes to be not expressed or disrupt vital ones. Iii) Researchers have developed technology to target
desired genes to specific sites within the animal’s germ line using homologous DNA recombination. Iv) This technique can replace
the endogenous gene with a truncated, mutated, or altered form, or completely replace it with a DNA sequence of choice. V)
Knock-in technology can determine when and where the promoter for a specific gene is activated, such as engineering a gene for
a fluorescent protein to glow green when activated.

🌑one method for the generation of knockout mice using homologous DNA recombination: Isolation and culture of embryonic stem
(ES) cells from the inner cell mass of a mouse blastocyst. Generation of the desired, altered form of the gene, bounded by suffi cient
DNA sequence from the native gene to facilitate homologous recombination. Introduction of the desired gene into the cultured ES
cells and selection of homologous recombinant cells in which the gene of interest has been incorporated. Sensitive polymerase chain
reaction (PCR) techniques can be used to determine which ES cell colonies have incorporated the desired gene into the correct
location. Injection of homologous recombinant ES cells into a recipient mouse blastocyst and surgical implantation of the blastocyst
into a pseudopregnant mouse. Mating of chimeric off spring heterozygous for the disrupted gene to produce homozygous knockout
mice.

Animal Models for Autoimmune Diseases

1)New Zealand Black (NZB): Systemic lupus erythematosus (SLE) is a systemic autoimmune disease characterized by fever, weakness,
arthritis, skin rashes, pleurisy, and kidney dysfunction. Affected individuals may produce auto-antibodies to various tissue antigens,
such as DNA, histones, RBCs, platelets, leukocytes, and clotting factors. The interaction of these auto-antibodies with their specific
antigens produces various symptoms, such as complement-mediated lysis, hemolytic anemia, and thrombocytopenia. NZB mice
spontaneously develop autoimmune hemolytic anemia between 2 and 4 months of age, detecting various auto-antibodies. F1 hybrid
animals develop glomerulonephritis from immune-complex deposits in the kidney and die prematurely by 18 months. SLE develops in
a mouse strain called MRL/lpr/lpr, which is homozygous for a defective fas gene called lpr. In absence of fas, mature peripheral T cells
continue to proliferate and produce cytokines, leading to grossly enlarged lymph nodes and spleen.

2) Non obese diabetic (NOD): A nonobese diabetic (NOD) mouse develops a form of diabetes resembling human insulin-dependent
diabetes mellitus (IDDM). The disease begins with lymphocytic infiltration into the pancreas’ islets and is strongly associated with
certain MHC alleles. Experiments show that T cells from diabetic mice can transfer diabetes to nondiabetic recipients. For
instance, when the immune system of normal mice is destroyed by x-rays and then reconstituted with bone-marrow cells from
NOD mice, the reconstituted mice develop diabetes. Conversely, when the immune system of healthy NOD mice is destroyed and
reconstituted with normal bone-marrow cells, they do not develop diabetes.
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3) Obese-strain chickens: Hypothyroidism is a condition characterized by the formation of autoantibodies and sensitized TH1 cells,
intense infiltration of the thyroid gland by lymphocytes, macrophages, and plasma cells, and an inflammatory response causing a
goiter. Antibodies are formed to thyroid proteins like thyroglobulin and thyroid peroxidase, which are involved in iodine uptake.
Binding these auto-antibodies to these proteins disrupts iodine uptake, leading to decreased thyroid hormone production.

4) Myasthenia gravis: Myasthenia gravis is an autoimmune disease caused by blocking antibodies. Patients produce auto-antibodies
that bind acetylcholine receptors on muscle motor end-plates, causing a progressive weakening of skeletal muscles and
ultimately destroying the cells bearing these receptors. Early signs include drooping eyelids and snarling. Without treatment,
muscle weakening can lead to severe eating and movement impairments. However, with appropriate treatment, the disease can
be managed well and afflicted individuals can lead a normal life. Treatment is crucial for managing this disease effectively.


5) Experimental autoimmune encephalomyelitis (EAE): Experimental autoimmune encephalomyelitis (EAE) is a well-studied animal
model that enhances understanding of autoimmunity. It is mediated by T cells and can be induced by immunization with myelin
basic protein (MBP) or proteolipid protein (PLP) in Freund’s adjuvant. Within 2-3 weeks, the animals develop cellular infiltration
of the myelin sheaths, leading to demyelination and paralysis. Most die, but some develop milder symptoms. Some develop a
chronic form resembling human MS. Those who recover are resistant to disease development from subsequent injections of
MBP and adjuvant.

6) Experimental autoimmune thyroiditis (EAT): and autoimmune arthritis (AA) are two conditions induced by immunization with
thyroglobulin in Freund’s adjuvant. EAT mimics Hashimoto’s thyroiditis, while AA is induced by immunizing rats with
Mycobacterium tuberculosis in Freund’s adjuvant, resulting in an arthritis similar to rheumatoid arthritis in humans. Both
conditions are induced by self-antigens immunization.

7) Rheumatoid arthritis: is an autoimmune disorder affecting women aged 40-60, causing chronic joint inflammation. The disease
affects the hematologic, cardiovascular, and respiratory systems. Individuals with rheumatoid arthritis produce rheumatoid
factors, which are auto-antibodies that react with IgG determinants in the Fc region. The classic rheumatoid factor is an IgM
antibody with this reactivity, binding to normal IgG, forming IgM-IgG complexes in joints.

Animal Models of Immunodeficiency Have been used to Study Basic Immune Function
1) Nude (Athymic) Mice: Nude mice, also known as nu/nu, are hairless and have a vestigial thymus, a trait controlled by a recessive
gene on chromosome 11. The mutated gene FOXN1 encodes a transcription factor that plays a role in cell differentiation and
survival. These mice are used in transplantation immunology studies due to their ability to tolerate allograft and xenograft tissue
from another species. Heterozygotic nu/wt littermates have hair and a normal thymus.

2) SCID Mouse (severe combined immunodeficiency): In 1983, Melvin and Gayle Bosma discovered an autosomal recessive mutation
in mice that led to a severe deficiency in mature lymphocytes. The SCID mouse had early B- and T-lineage cells but a virtual
absence of lymphoid cells in the thymus, spleen, lymph nodes, and gut tissue. Precursor cells in the SCID mouse were unable to
differentiate into mature functional B and T lymphocytes, leading to infection. Hematopoietic cells other than lymphocytes
developed normally in the SCID mouse. The mutation was found in a gene called protein kinase, DNA activated, catalytic
polypeptide (PRKDC), which participates in the double-stranded DNA break-repair pathway. This defect is a leaky mutation,
suggesting components of both humoral and adaptive immunity.

3) RAG Knockout Mice: The deletion of recombination-activating enzymes (RAG-1 and RAG-2) in RAG knockout mice results in a
defect in both B and T cells, preventing them from rearrangement of immunoglobulin or T-cell receptor genes. This leads to
abnormal development, and cells with abnormal rearrangements are eliminated in vivo, resulting in the absence of both B and T
cells from the lymphoid organs.

In vitro system: Quantification of cytokines- 1) ELISA assay: i) ELISA is an enzyme-linked immunosorbent assay (ELISA) used to detect
and measure antibodies, hormones, peptides, and proteins in blood. Ii) It works by detecting the presence and quantity of antigens
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binding. Iii) To increase sensitivity and precision, plates must be coated with high affinity antibodies. Iv) ELISA tests can be classified
into three types: indirect ELISA, sandwich ELISA, and competitive ELISA.
1) Indirect ELISA: i) detects the presence of an antibody in a sample by attaching the antigen to the wells of a microtitre plate. Ii) A
sample containing the antibodies is added to the antigen-coated wells for binding. Iii) The free primary antibodies are washed away,
and the antigen-antibody complex is detected by adding a secondary antibody conjugated with an enzyme that can bind with the
primary antibody. Iv) The absorbance of the coloured product is measured by spectrophotometry.
2) Sandwich ELISA: i) detects the presence of an antigen in a sample by coating the microtitre well with the antibody and adding an
enzyme-linked secondary antibody that binds to another epitope on the antigen. Ii) The enzyme-specific substrate is added to the
plate to form a coloured product.
3) Competitive ELISA: i) detects antigen concentration in a sample by coating the microtitre wells with the antigen and incubating the
antibodies in a solution with the antigen. Ii) The enzyme-linked secondary antibody is added to detect the number of primary
antibodies present in the well, and the concentration is determined by spectrophotometry.

🌑 Process: i) Assay plate coated with anti-antibody (for a particular cytokine]. This antibody called capture antibody. Ii) suspension of
the cell population under investigation is then added to the coated plates and incubated with any appropriate stimulating agents. Iii)
The cells settle onto the surface of the plate, and any interferon that is secreted by the stimulated cells is bound by the capture
antibodies on the plate, creating a ring of IFN- antibody complexes around each interferon-producing cell. Iv) The plate is then washed
to remove the cells, and an enzyme- linked antibody, the “detection antibody,” specific for an antigenic determinant on IFN different
from that bound by the capture antibody, is added and allowed to bind. V) After another washing step, an ELISPOT substrate is added.
Vi) Substrates for ELISPOT assays are normally colorless but, when acted on by their cognate enzyme, they precipitate out of solution,
leaving a clearly demarcated, colored “spot” wherever the enzyme-conjugated antibody bound. Vi) Types Of ELISA: i) Indirect ELISA –
Antigen is coated to the microtiter well. Ii) Sandwich ELISA – Antibody is coated on the microtiter well. Iii) Competitive ELISA –
Microtiter well which is antigen-coated is filled with the antigen-antibody mixture.

🌑Applications: i) The presence of antibodies and antigens in a sample can be determined. Ii) It is used in the food industry to detect
any food allergens present. Iii) To determine the concentration of serum antibody in a virus test. Iv) During a disease outbreak, to
evaluate the spread of the disease, e.g. during recent COVID-19 outbreak, rapid testing kits are being used to determine presence of
antibodies in the blood sample.

🌑 Advantages: i) Results fetched from ELISA gives an accurate diagnosis of a particular disease since two antibodies are used. Ii) Can be
carried out for complex samples as the antigen is not required to get purified to detect. Iii) It is highly responsive since direct and
indirect analysis methods can be carried out. Iv) It is a rapid test, yields results quickly. V) Possible detection for ELISA ranges from the
quantitative, semi-quantitative, standard curve, qualitative, calibration curve models etc. Vi) Easier to perform and uncomplicated
process as compared to other assays which require the presence of radioactive materials.

2) Functional assay of phagocytosis:

🌑Phagocytosis Assay: kill fluorescein-labeled Candida albicans as the target for ingestion Inhibiting phagocytosis in control tube by
adding cytochalasin B After phagocytosis is complete, EB Ethidium Bromide (50 pg/ml) is added to label the external organisms On
excitation at 488 nm, the internalized bacteria fluoresce green (fluorescein), while the sure face-attached organisms are red This
occurs because of the phenomenon of resonance energy transfer between FITC and EB. The EB stains the nuclear material of the
external bacteria; because these organisms are also labeled with FITC, the FITC excites the EB, resulting in red fluorescence.
Internalized organisms are not exposed to EB and so cannot emit red fluorescence.

🌑Oxidative metabolism: involves the formation of reactive oxygen species, such as O2-, H202, and OH-, through various intracellular
sources such as mitochondrial oxidation, the microsomal cytochrome P-450 system, and plasma membrane NADPH oxidases.
Neutrophils also serve as scavengers of free radicals, including superoxide dismutase, catalase, glutathione peroxidase, cytochrome c
oxidase, glutathione, ascorbate, cu-tocopherol, p-carotene, and polyunsaturated fatty acids.

🌑Hydrogen peroxide Production assay: The the nonfluorescent molecule 2’7’-dichlorofluorescin diacetate (DCFH-DA) is loaded into the
cells acting as oxidizable substrate DCFH-DA is non-polar and can pass through the plasma membrane inside the cell Inside the cell,
the enzyme esterase deacetylate it to form polar DCFH which is retained in the cytoplasm and myeloperoxidase positive intracellular
granules The peroxidases oxidizes DCFH to fluorescent DCF (2’,7’Mdichlorofluorescein (DCF) whose green fluorescence at 525 nm is
easily measurable on the flow cytometer. The amount of DCF formed is proportional to the cellular oxidant production (H2O2)
 11

🌑 Production: The study uses flow cytometry to measure intracellular superoxide anion (OJ) production in stimulated neutrophils using
hydroethidine (HE). This method, when combined with DCFH-DA, can provide a comprehensive assessment of the oxidative burst or
determine the amount of superoxide formed within cells.