California
Association
for
Medical Laboratory Technology
Distance Learning Program
| An
Overview of the Immune System, Part Two: Author: © California Association
for Medical Laboratory Technology. CAMLT is approved by the California Department of Health
Services 1895 Mowry Ave, Suite 112 Notification of Distance Learning Deadline This is a reminder that all the continuing
education units required to renew your license must be earned no later
than the expiration date printed on your license. If some of your
units are made up of Distance Learning courses, please allow yourself
enough time to retake the test in the event you do not pass |
| This course is configured to be completed on-line. You can register
for the course, submit secure payment using a credit card via PayPal,
take the quiz on-line and receive your graded score.
If you pass, your certificate will be mailed to you from
the CAMLT office. If you fail, you must submit new payment and obtain a new PayPal receipt each time you take the test. A certificate will be issued only if you have paid for re-taking the course and you pass the test. If you want to submit your registration and quiz via fax or mail you should print the Adobe Acrobat version of the course which includes the required Registration/Quiz form. |
| Links to: On-line REGISTRATION, PAYMENT and QUIZ Printable Acrobat version of this course * Review Questions at the end of this Course Other Distance Learning Courses |
|
An Overview
of the Immune System, Part Two:
The Generation & Evaluation of Immune Responses
ABSTRACT
Although immunology is a complex and specialized area of medicine
and laboratory science, a basic understanding of how normal immune responses
occur can be helpful to anyone in the health care field. In the first course
of this two-part series, The Cells and Cell Surface Molecules of the Immune
System, the basic components of the immune system were reviewed. Here,
in the second course, the characteristics of antigen-specific immune responses
will be introduced, and the cellular interactions necessary for normal humoral
and cell-mediated immune responses will be presented. In addition, techniques
that are utilized to assess the immune system and/or to utilize immunologic
techniques in the clinical laboratory will be reviewed.
OBJECTIVES
Upon completion of this course, the reader will be able to:
• List the four hallmarks of antigen-specific immune responses
• Identify which innate white blood cells can serve as antigen-presenting
cells in adaptive immune responses
• Match the appropriate class of MHC molecule to the T cell subset that
requires it for antigen presentation
• Describe the role of CD4-expressing T helper cells in humoral and cell-mediated
antigen-specific responses
• List one clinical laboratory technique that enumerates lymphocyte subsets,
and at least one additional technique that provides information on immune system
function
Preface/Author’s Note
This two-part series of immunology courses is dedicated to the
memory of Dr. Janis Kuby. Dr. Kuby was an exceptional professor of immunology
at San Francisco State University, and the original author of Kuby Immunology,
one of the most widely-used immunology textbooks throughout the country. Janis
Kuby will never be forgotten by anyone who has ever had the benefit of her knowledge
and love of immunology, either through her classroom teaching or her outstanding
textbook.
LIST OF FREQUENTLY-USED ABBREVIATIONS WBC - white blood cells Ig - immunoglobulin; also known as antibody TCR - T cell receptor MHC - major histocompatibility complex CTL - cytotoxic T lymphocyte (CD8-positive T cell) TH - T helper cell (CD4-positive T cell) APC - antigen-presenting cell DC - dendritic cell IL-2 - interleukin-2 CTL-P - mature CTL precursor PE - phycoerythrin FITC - fluorescein isothiocyanate ELISA - enzyme-linked immunosorbent assay
INTRODUCTION
The immune system is constantly challenged to protect individuals
from the enormous array of foreign materials to which they are exposed. The
protection provided by the human immune system is a collaborative one, calling
on both innate immunity, which is present in some form in all multicellular
plants and animals, and adaptive immunity, which is a relatively recent product
of evolution found in more advanced vertebrates (1). As reviewed in a previous
course (2), the innate immune system prevents foreign materials, known as antigens,
from gaining entry to the body through a variety of physical and chemical barriers.
If a toxin, foreign protein, pathogen, or any other antigen succeeds in breaching
these barriers, it would soon encounter a host of innate system cells and cellular
defenses capable of preventing infection and/or eliminating pathogens and antigens
from the body. While these cellular defenses can recognize and respond to foreign
antigens, the innate immune system is relatively non-specific, i.e., it is not
sufficiently specialized to discriminate between different types of foreign
molecules. Although it is a highly-effective first line of defense against most
infections and toxic exposures, the innate system does not always succeed in
preventing disease. When disease is the outcome, the innate immune response
serves as the critical first step in generating a more sophisticated and exquisitely
specific adaptive, or acquired, immune response. Adaptive immunity is designed
to recognize and eliminate a particular antigen in a very specific fashion,
and to remember the antigen so as to be able to protect against it in the future.
It is this combination of initial adaptive response and development of antigen-specific
memory which is responsible for immunity as we know it.
Immunity is defined as “ the state of protection from infectious
disease” (1). The concept is an ancient one, dating back to an epidemic,
“The Plague of Athens,” in 430 BC, when an historian (Thucydides)
observed that only those who had recovered from the disease could nurse the
sick without becoming sick again themselves. The root of the word immunity is
the Latin immunis, meaning “exempt,” reflecting the early
understanding of exemption or protection from recurrent disease. Over the centuries,
especially during the twentieth century and into the twenty-first century, scientists’
understanding of how the immune system accomplishes this feat has become more
and more detailed. The primary goal of this course is to describe how the different
types of white blood cells (WBC) interact with antigen and with each other to
bring about adaptive antigen-specific immunity. In addition, the course will
provide an overview of some of the specialized ways in which the status of the
immune system can be evaluated in the clinical laboratory.
I. ADAPTIVE IMMUNE RESPONSES
The WBC of the immune system and the function(s) of each cell type
have been described in the first part of this series (2). They include granulocytes
such as neutrophils, and mononuclear cells such as monocytes/macrophages, natural
killer cells, and lymphocytes. It is the ability of B and T lymphocytes to specifically
recognize and remember individual antigens that makes antigen-specific adaptive
immune responses possible (1). These cell types are capable of antigen recognition
due to the presence of antigen receptors on their cell surfaces. B lymphocytes
(also known as B cells) utilize surface-bound antibody or immunoglobulin (Ig)
molecules as antigen receptors. T lymphocytes (or T cells) use a different,
but antibody-like molecule called the T cell receptor (TCR). Surface Ig and
TCR are proteins that are anchored in the cell membrane, and have an antigen-binding
“pocket” with a particular three-dimensional shape extending outward.
When a cell encounters an antigen that has the proper complementary shape to
fit into the pocket (like a key fitting into a lock), the antigen will bind
to the receptor molecule in a very specific fashion. The interaction of antigen
and receptor on the cell surface initiates a signaling cascade that turns on
new gene expression in the nucleus, and ultimately, leads to the activation
of the T or B cell.
Adaptive, antigen-specific immune responses share four distinguishing
characteristics which set them apart from innate, non-specific responses (1).
All of these characteristics are mediated by the antigen receptors on B and
T cells. They are antigenic specificity, diversity, self/non-self recognition,
and immunologic memory.
Antigenic specificity permits the immune system to distinguish
differences among antigens. Many antigens differ greatly, making it easy for
the immune system to discriminate among them. However, the immune system has
such exquisite specificity that it can also discriminate between antigens that
differ only by a single amino acid. The specificity of the immune system lies
in the antigen receptors on B and T cells. On the surface of a single T or B
cell, there are thousands of TCR or Ig molecules. However, on a single cell,
every antigen receptor is identical, with the same antigen-binding pocket that
is complementary to a specific antigen. This ensures that a given T or B cell
will recognize only a certain shape on an antigen, and as long as that antigen
does not change its shape, will continue to recognize the same antigen time
and time again.
Diversity is the ability of the immune system to generate
a tremendous number of different antigen receptors, allowing it to recognize
millions of unique sites on antigens. Although each individual B or T cell recognizes
only one particular shape on an antigen, collectively all of the B and T cells
together have the capacity to recognize more than one hundred million different
antigen sites. This is accomplished by an extraordinary system of DNA rearrangements
that is unique to B and T cells. By physically cutting, moving, and pasting
small portions of the Ig or TCR genes into new combinations, the immune system
is capable of generating enormous numbers of different B and T cells from a
relatively small amount of genetic material. Each cell thus has a cell surface
antigen receptor with a slightly different antigen-binding pocket. This means
of generating specificity and diversity through genetic recombination was unprecedented
in biology when it was first described for Ig genes in 1976 by S. Tonegawa.
Tonegawa’s work revolutionized the field of immunology, and the importance
of his contribution was recognized with a Nobel prize in 1987. It took until
the 1980s for scientists to identify TCR genes, which proved to have similar
structure and undergo the same genetic rearrangement as Ig genes. While the
structure of Ig and TCR genes and the highly ordered manner in which they rearrange
is well-established, there are some aspects of antigen-receptor generation (such
as some details of the signals and enzymes involved in the cutting and pasting
of DNA) that remain to be elucidated. The story of immune system gene rearrangements
continues to be a fascinating one, and can be read about in detail in reference
1 and many other immunology and genetics texts.
Self/non-self recognition is the ability of the immune
system to distinguish self components from non-self (foreign) antigens and cells.
This ensures that the immune system mounts responses only to non-self antigens
or cells, preventing (under normal circumstances) autoimmune responses. Self/non-self
recognition of cells is carried out by the TCR on T cells, and utilizes the
major histocompatibility complex (MHC) molecules. These molecules, also known
in humans as Human Leukocyte Antigens (HLA) or the transplantation antigens,
are identical on the cells within a single individual, but differ from individual
to individual. There are two major classes of MHC molecules, class I (HLA-A,
B, C) and class II (HLA-D), that are utilized in different types of immune cell
interactions (as described below). Discrimination between self and foreign antigens
by B and T cells is carried out by their respective antigen receptors through
a process known as tolerance that occurs during lymphocyte development.
Immunologic memory is the phenomenon that gives rise to
rapid and protective antigen-specific immunity upon a second or subsequent encounter
with the same antigen. As a result of a first-time exposure to a specific antigen
(i.e., a particular pathogen), the adaptive immune system mounts a specific
primary response targeted against that antigen. This response tends to be slow
to develop (7-10 days to peak response in the case of an antibody response),
and is of limited magnitude and duration (1). If the faster-acting but relatively
non-specific innate immune response is unable to eliminate a pathogen, an exposed
individual typically gets sick the first time he or she encounters a pathogen.
In other words, the pathogen wins the race the first time around because the
innate response is insufficient and the primary adaptive response is slow. However,
during the primary adaptive response, a pool of antigen-specific memory B and
T cells is produced which may persist for years or even decades. These memory
cells are primed to respond to a particular antigen, and so when they encounter
the identical antigen a second time, they are able to respond much more rapidly
(3-5 days for peak antibody response), more vigorously (100-1000 times higher
peak antibody levels), and for a longer period of time. Memory cells can be
described as fulfilling the Olympic motto: “Citius, Altius, Fortius”
(faster, higher, stronger)! It is this olympian effort that enables the immune
system to win the race over a pathogen during a memory or secondary response,
as it can swiftly and effectively contain and eliminate the pathogen before
the onset of disease. It is immunologic memory, which is a function of the specificity
of B and T cells, that enables the development of specific immunity as a result
of natural exposures to pathogens. Vaccination takes advantage of this characteristic
of antigen-specific immunity by replacing the natural primary exposure to a
pathogen with exposure to a harmless form of the same antigen(s). Therefore,
upon the first exposure of a vaccinated individual to the actual pathogen, an
effective (and hopefully protective) secondary response is generated.
The immune system encounters antigens within the body in two forms.
Soluble antigens are found floating freely in the liquid portion of the blood
and in the lymph fluid that drains the tissues and filters through lymph nodes.
Examples of soluble antigens include bacteria introduced through a break in
the skin, or a bacterial protein product like diphtheria or tetanus toxin. Cell-associated
antigens are foreign proteins or altered self proteins that have been synthesized
within a cell, such as viral proteins in virally-infected cells, antigens made
by intracellular bacteria, and tumor-associated mutant self proteins on cancerous
cells. In order to effectively deal with both soluble and cell-associated antigens,
the adaptive immune system can mount two distinctly different types of antigen-specific
responses.
The humoral immune response, mediated by antibodies, has evolved
to deal with soluble antigens. The term “humoral” refers to body
fluids, which were known as “humors” in the late 1800s when it was
demonstrated that immunity could be transferred via fluids such as serum (1).
The surface Ig that acts as the antigen receptor on B cells can bind soluble
antigens, which sets off a chain of events (described in more detail below)
that eventually gives rise to antibody-secreting plasma cells. The secreted
antibody, which circulates in the blood and lymph, binds to the specific soluble
antigen that stimulated the response. The resulting antibody-antigen complex
can be cleared by the kidneys, lysed by the complement cascade, and/or removed
by phagocytic cells such as monocytes and macrophages. In the case of biologically
active antigens like toxins or free floating bacteria or viruses, the binding
of antibody may neutralize the antigen, i.e., prevent it from interacting with
cells, thus preventing toxic effects or infection.
Cell-associated antigens are the product of cells that are defective in some
way (mutated and/or cancerous), infected by intracellular organisms (viruses,
intracellular bacteria, some parasites), or foreign cells that have entered
the body. Mutated self antigens or foreign antigens produced within a cell are
digested into small pieces, which become physically associated with self MHC
molecules also produced within the cell. The digested antigen-MHC complex then
migrates to the cell surface, where it could be detected by the adaptive immune
system. In most cases, the digested antigen on the cell surface cannot be removed
by circulating antibodies. More importantly, if the antigen is a product of
an intracellular pathogen, the pathogen itself is sheltered within the cell,
beyond the reach of the humoral immune response. It is necessary, therefore,
for the immune system to be able to rid the body of a defective, infected, or
foreign cell as a whole. The antigen-specific cell-mediated or cytotoxic response,
which is capable of killing whole cells by inducing cell lysis, has evolved
to meet this need. Adaptive cytotoxic immune responses are carried out by CD8-expressing
cytotoxic T lymphocytes (CTL), as described below.
II. THE COOPERATION OF INNATE AND ADAPTIVE IMMUNE RESPONSES
In a healthy immunocompetent individual, i.e., a person with intact,
properly functioning innate and adaptive immune systems, antigen-specific immune
responses are generated as a result of a series of interactions between different
types of WBC. A schematic illustration of these interactions is shown in Figure
1. Although humoral responses are ultimately carried out by antibody-secreting
B cells, and cell-mediated cytotoxic responses are executed by CTLs, both types
of responses require the same collaborative first step. The critical cell types
in this initial step are antigen-presenting cells (APCs) and the CD4-expressing
subset of T cells known as T helper (TH) cells.
In the example illustrated in Figure 1, the
antigen (represented by asterisks) is found on a virus, which can be both free-floating
in body fluids (when first entering the body as well as when released by infected
cells), and located inside infected cells (the rectangular cells along the left
side of the figure). In order to initiate an adaptive immune response, it is
necessary for this viral antigen to be recognized by TH cells. Unlike
B cells, T cells cannot recognize free-floating or soluble antigens. Rather,
the TCR on a T cell recognizes digested antigen on the surface of another cell,
but only when the antigen is part of an antigen-MHC complex. As previously described
(1, 2), there are two classes of MHC molecules: Class I, which is expressed
on virtually all cells throughout the body, and Class II, which is expressed
only on select cells within the immune system. When cell-associated antigens
are produced within any type of cell (such as the virally-infected cells in
Figure 1), the digested antigens are complexed with MHC Class I molecules. However,
the initiation of either a humoral or cytotoxic adaptive immune response depends
on the ability of TH cells to recognize antigen plus MHC Class II
molecules. This critical recognition event requires the participation of APCs,
most of which belong to the innate immune system.
Since the expression of MHC Class II molecules is strictly controlled,
only certain immune system cells can act as APCs for TH cells. An APC is capable
of taking up virtually any protein antigen from outside the cell, digesting
it, and displaying an antigen-MHC Class II complex on its surface for interaction
with TH cells. The most effective APCs for interacting with and stimulating TH cells in a primary immune response are dendritic cells (DC). DCs are innate,
macrophage-like cells, usually found in tissues and/or lymph nodes, that collect
antigens, digest them, and present them to T cells with a high concentration
of MHC Class II molecules. Other innate APCs are monocytes (circulating in the
blood) and macrophages (monocytes that have moved into tissue), which become
activated after phagocytosis of antigen and express MHC Class II molecules.
The final type of APC that can interact with TH cells is B cells, which can
capture and internalize antigen via cell surface Ig antigen receptors, and then
process and present antigen-MHC Class II complexes on the cell surface.
In Figure 1, an APC that has encountered some free-floating viruses
is shown at the top of the figure. In this example, the APC could be a DC, macrophage,
or monocyte, using innate Toll-like receptors for the recognition of pathogens
(1, 2). Upon interaction with the viral particles, the APC becomes activated,
internalizes and processes the virus particles, and presents viral antigens
plus MHC Class II to the TH cell shown to its right. This illustrates how innate
system cells serve as a bridge to the initation of adaptive responses, i.e.,
facilitating the antigen-specific interaction of the TCR on a TH cell with antigen
plus MHC Class II. It is important to remember that this particular APC will
not simply interact with the first TH cell it encounters. Rather, it will be
able to interact only with those TH cells that carry a TCR with an antigen-binding
pocket that is a match for the relevant viral antigens. In many cases, APC-TH
cell interactions will occur in lymph nodes and other lymphoid tissues where
antigens are trapped and concentrations of APCs (especially DCs) and T cells
are high, maximizing the likelihood of a successful match between antigen and
its antigen-specific T cell.
The specific binding of the TCR to the viral antigen-MHC Class
II complex on the APC sends a signal to the nucleus of the TH cell. Although
not shown in Figure 1, all APCs interacting with T cells must also provide an
additional stimulatory signal via cell-to-cell contact with the TH cell in order
to complete the activation of the T cell. When a TH cell receives the correct
combination of signals from the TCR/antigen/MHC Class II interaction plus cell-to-cell
contact, it becomes fully activated, capable of driving all other aspects of
adaptive immune responses.
One of the major functions of antigen-activated TH cells in immune responses
is the secretion of cytokines. Cytokines are secreted proteins which send signals
or messages between cells. They were first described as messenger molecules
involved in signaling between different types of WBC (also known as leukocytes),
and so some cytokines bear the name “interleukin,” meaning “between
white cells.” Cytokines are produced by one cell in response to a stimulus,
and then, depending on the cytokine, act on the same cell or adjacent cells
to influence their behavior. Cytokines are produced by both innate and adaptive
immune system cells, and are very important in both types of responses, but
for the purposes of this course, will be discussed only in the context of adaptive
responses. As shown in Figure 1, cytokines act at a variety of steps throughout
both humoral and cytotoxic immune responses.
The newly-activated TH cell at the top of Figure 1 begins to make an essential
cytokine, originally known as T cell growth factor, but now known as interleukin-2
(IL-2). IL-2 produced by an activated TH cell can act on itself, as well as
on other T cells. IL-2 is absolutely required for the antigen-activated TH cell
to divide and proliferate. This proliferation dramatically increases the number
of antigen-specific activated TH cells, which is necessary to ensure an immune
response of sufficient magnitude. (It also contributes to the swelling and tenderness
of lymph nodes during an acute immune response.) In addition to proliferation,
the TH cells begin the process of differentiation, in which cells fully mature
and/or acquire new function. In the case of TH cells, differentiation allows
the production of a variety of cytokines in addition to IL-2. It also permits
the expression of additional cell surface molecules that will deliver cell-to-cell
signals, and the development of a pool of antigen-specific memory TH cells.
It is through the secretion of cytokines, as well as providing co-stimulatory
signals, that TH cells “help” both antibody- and cell-mediated immune
responses to develop.
III. GENERATION OF AN ANTIBODY RESPONSE
In Figure 1, while an innate APC is processing and presenting soluble
viral antigens, a B cell that is specific for the same viral antigens is recognizing
and binding to free-floating virus particles. B cells are capable of recognizing
soluble antigens prior to processing, and more importantly, without the participation
of MHC molecules. The B cell binds to the virus particles using the surface
Ig molecules which serve as the B cell receptors for antigen. Similar to TCR
binding, binding of antigen to B cell receptors delivers a signal to the nucleus
of the B cell, initiating a cellular activation process. Back at the cell surface,
the bound virus particle and Ig molecule are internalized by the B cell and
processed in much the same way as in an innate APC, resulting in the digested
viral antigen plus MHC Class II being displayed on the B cell surface. The B
cell is then ready to interact with one of the newly-generated activated TH
cells that is specific for the same antigen, in order to complete the activation
process.
When a B cell displaying viral antigen plus MHC Class II encounters
an activated TH cell that recognizes the same viral antigen, the
two antigen-specific cells form a pair with the antigen serving as a bridge
between them (as shown on the right side of Figure 1). These antigen-specific
B-T cell pairs form in lymph nodes and other secondary lymphoid tissues, where
T cell-rich and B-cell rich zones meet. Cell-to-cell contact with an activated
TH cell, which ensures the proper delivery of co-stimulatory signals,
is required for the B cell to become fully activated. Upon activation, the B
cell expresses cytokine receptors that will enable it to receive cytokine signals
from the TH cell. The interaction also brings the TH cell
into close proximity to the B cell so that it can effectively secrete additional
cytokines to stimulate the B cell. These cytokines, which include IL-4, IL-5,
and IL-6, are collectively called “B cell stimulatory cytokines,”
and drive the activated B cell through the steps necessary to become an antibody-secreting
plasma cell.
As was seen with the TH cell, the newly-activated B cell responds
first to cytokine signals from the TH cell by proliferating in order
to increase the number of antigen-specific B cells. This B cell proliferation
also contributes to lymph node swelling during acute immune responses. Following
several rounds of B cell division, there will be a pool or clone of B cells,
all with the same antigen specificity as the original B cell that was activated
by antigen and the TH cell. Once the activated B cells have stopped
dividing, additional cytokine signals from the TH cell stimulate
them to differentiate.
Differentiation for B cells takes two paths. One path generates a pool of antigen-specific
memory B cells, some of which may persist for the life of an individual. It
is these memory B cells, in concert with memory TH cells, that will
facilitate the rapid, high quality secondary antibody response upon subsequent
exposure(s) to the identical antigen. The other path of B cell differentiation
results in the Ig molecule, which originally served as the cell-surface antigen
receptor, being actively secreted by the B cell. Fully differentiated B cells
that secrete Ig (also known as antibody) are known as plasma cells (shown at
the bottom right of Figure 1). Therefore, in the example shown, the very same
virus-specific Ig that was anchored in the surface of the B cell is now being
produced in a soluble form by a clone of plasma cells, to deal with free-floating
virus particles in body fluids. Secreted Ig circulates in the blood and lymph,
where it binds to the appropriate antigen and facilitates antigen clearance
and/or neutralizes its activity, i.e., preventing the virus particles from infecting
new cells.
IV. GENERATION OF A CELL-MEDIATED RESPONSE
In the case of a viral infection, humoral responses are effective
in blocking viral infection and eliminating free-floating virus particles. However,
cell-associated viral antigens and the virus itself within infected cells are
not easily eliminated by circulating antibody. Instead, such cells (and associated
antigens and pathogens) need to be eliminated by cell-mediated or cytotoxic
immune responses. While innate cell-mediated immunity, involving macrophages
and natural killer cells, serves as an important first line of defense (1, 2),
the remainder of this course will focus on adaptive cell-mediated immune responses,
which involve CD8-expressing T cells (CD8 T). Also known as cytotoxic T lymphocytes
(CTL), this subset of T cells is responsible for antigen-specific cytotoxicity,
which results in the lysis and death of whole cells in an antigen-specific manner.
CD8 T cells in the circulation or tissues are mature T cells that
carry antigen-specific TCR on their surfaces, but are not yet capable of killing.
An example of one of these cells, referred to as a CTL precursor (CTL-P), is
shown on the upper left of Figure 1. Like TH cells, the TCR on CTL-P
cells will recognize only digested antigen plus MHC molecules on the surface
of cells. However, in a very important difference from TH¬ cells,
CTL-P cells recognize antigen plus MHC class I molecules, which are found on
all nucleated cells in the body, including the APCs that also can facilitate
MHC Class II-associated antigen presentation. For the activation of a CTL-P,
the TCR on the CD8 T cell interacts with an antigen-MHC Class I complex, usually
presented by DCs or other APCs (as shown on the left side of the APC at the
top of Figure 1). APCs are necessary for the initial activation of CTL-P due
to a requirement for additional co-stimulatory signals. However, once activated
and fully differentiated, CD8-expressing CTLs can respond to antigen plus MHC
Class I on virtually any cell type in the body as described below.
The further activation of the CTL-P is driven by cytokines produced
by nearby antigen-activated TH cells. It is not clear whether a CTL-P
and a TH cell have to interact with the same APC at the same time,
or if a TH activated by a nearby APC will suffice (1). In either
case, an activated CTL-P cell expresses the receptor that will permit it to
respond to IL-2, the cytokine necessary for any T cell to proliferate. Activated
CD8 T cells make little or no IL-2, therefore they must rely on activated TH
cells to help them by providing IL-2. This illustrates once again the collaborative
nature of the innate and adaptive immune systems, and the critical role for
TH cells in both humoral and cytotoxic immune responses. Ultimately,
the help provided by TH cells to CTL-P and CTLs is similar to that
provided to B cells—the secretion of cytokines that provide proliferation
and differentiation signals. However, there is one major difference between
B and CD8 T cell help. In order to become fully activated, B cells require cell-to-cell
contact with an activated TH in addition to the secretion of cytokines.
In contrast, neither the CTL-P nor the CTL appears to require physical contact
with the TH cell, but depends instead on its secreted cytokine signals.
Upon receipt of the IL-2 signal from a TH, the activated CTL-P
proliferates, increasing the number of potential antigen-specific CTLs. As the
CTL-P cells proliferate, they continue to respond to IL-2 as well as to additional
cytokines secreted by TH and possibly even by APCs. These cytokine
signals allow the CTL-P cells to differentiate into fully-functional, lethal
CTLs, as well as into memory CTLs. All of these CTLs bear the same TCR as the
original CTL-P cell that was activated by viral antigen, but now have the ability
to kill and/or remember any cell bearing the same viral antigen.
All proteins manufactured within a cell show up on the cell surface
in digested form as an antigen-MHC Class I complex, regardless of whether the
proteins are normal self, mutated self, or foreign. This process occurs in nearly
all cells in the body, but the levels of MHC Class I expression vary among cell
types, with the highest levels of expression on lymphocytes. CD8 T cells develop
tolerance to self antigens during immune system development, and under normal
circumstances, do not respond to self-antigen plus self-MHC complexes. However,
when foreign proteins are being synthesized inside a pathogen-infected cell,
or mutant proteins are being produced that are perceived as foreign, it is the
combination of foreign antigen plus self-MHC Class I that successfully engages
the TCR of a fully-mature CTL and initiates cell killing. In Figure 1, some
of the virally-infected cells along the left side of the figure are shown as
expressing viral antigen plus MHC Class I on their surfaces. They are probably
not immune system cells, do not express MHC Class II, and so cannot act as APC
for CD4+ TH cells or CTL-P cells. They are, however, poised to serve
as targets for fully-functional CTLs.
As illustrated on the left side of Figure 1, a CTL utilizes its
TCR to bind to a target cell in an antigen-specific fashion. Once it has bound
to its target via cell-to-cell interactions, the CTL usually uses one of two
ways to kill the offending cell. It can secrete a pore-forming protein known
as perforin, that inserts itself in the membrane of the target cell and permits
the delivery of toxic granules into the target cell. Within five minutes of
contact with a CTL, the contents of the granules begin to induce target cell
death through a suicide process known as apoptosis. Alternatively, in the absence
of perforin, CTLs can induce apoptosis via an interaction between two membrane
proteins, Fas and Fas ligand, on the surfaces of the target cell and CTL, respectively.
After either one of these interactions, the CTL releases the mortally-wounded
target cell (shown as a hatched cell in Figure 1), leaving it to die and ultimately
break apart within a few hours or less. Interestingly, the CTL is resistant
to its own means of killing other cells, so this killing can be repeated again
and again. Therefore, not only does the original CTL precursor proliferate to
increase the number of antigen-specific CTLs, but each of those CTLs is capable
of killing many target cells. Upon the death of target cells, pathogens (such
as more viruses) and/or soluble antigens may be released into the circulation.
If cell-mediated responses occurred in the absence of humoral responses, this
release of pathogens and antigen might be more harmful than helpful. However,
many pathogens and antigens are soluble at some point in their journeys through
the body, and so may have stimulated both humoral and cell-mediated responses.
Then, upon the death of infected cells as a result of CTL activity, newly-released
organisms and antigens could be bound and cleared by circulating antibody being
produced concomitantly by plasma cells.
V. IMMUNOLOGIC ASSESSMENTS
In the first part of this series, the cells and cell surface molecules
that make up the immune system were reviewed (2). In this course, the cellular
interactions necessary to generate antigen-specific responses that battle primary
exposures and ensure long-term immunity have been presented. The course will
conclude with an examination of some of the laboratory assays that are used
to evaluate the immune system as well as a discussion of immunologic techniques
(3). In particular, the ability of flow cytometry (Figure 2) and ELISA testing
(Figure 3) to contribute information about the immune system will be emphasized.
Although immunology laboratories and specialized immunologic techniques are
often found only in large institutions and/or reference laboratories, such techniques
are becoming more common in many areas of the routine clinical laboratory. Even
if a clinical laboratory does not perform any of these assays, clinical scientists
at all levels may be called upon to facilitate the performance of such testing.
It may be helpful, therefore, to be familiar with some of these techniques.
A traditional method of testing immune cell function is the delayed-type
hypersensitivity skin test, where a small dose of antigen is introduced intradermally,
then the injection site is observed for 24-48 hours for evidence of a localized
immune response (3). Health care workers are familiar with this type of test
being used to determine whether or not an individual has been previously exposed
to a particular antigen or pathogen, such as the purified protein derivative
(PPD) skin test for tuberculosis. However, skin testing is also used clinically
to evaluate the ability of an individual’s immune system to make a cell-mediated
memory response to common antigens. Such a response requires many of the cellular
interactions shown in Figure 1, and so can provide an indication of the overall
status of the immune system. Therefore, in situations where the immune system
is likely to be suppressed, (i.e., a person with HIV infection, undergoing cancer
treatment, or receiving an organ transplant), the ability of an individual to
respond to one or more commonly-encountered antigens in a skin test can be a
valuable means of assessing overall immunocompetence (3). The information provided
by skin testing is limited due to its qualitative nature, i.e., it can only
show if an individual does or does not respond. There are other, more quantitative
means of evaluating the immune system, some of which can be performed in the
typical clinical laboratory, and others that are likely to be performed only
in highly specialized hospital, research, or reference laboratories.
Physicians have long relied on the hematology laboratory and the
complete blood count to provide them with critical information regarding the
status of the immune system. The total WBC count and the white cell differential
count (whether manual or automated) provide insight into the possible presence
or absence of infection and/or immunologic disorders. The serology laboratory
can provide additional clues, ranging from quantitative measures of serum immunoglobulin
levels to more specific diagnostic information such as antibody titers against
a particular pathogen. However, as the practice of medicine has become more
sophisticated in its understanding of the immune system, it has been necessary
for the clinical laboratory to provide more detailed immunologic analyses. Out
of this need has evolved the field of flow cytometry, which has taken the best
and brightest technology that hematology has to offer, and coupled it with the
powerful specificity of antigen-antibody reactions of the serology lab.
Flow cytometry permits the discrimination of subsets of white blood
cells, especially lymphocytes, that cannot be distinguished simply on the basis
of morphology under a light microscope or in an automated hematology analyzer
(3). As our understanding of the different roles played by the TH, CTL, and
B cells has increased, so has the need increased to be able to enumerate these
lymphocyte subsets in much the same way that a white cell differential enumerates
neutrophils, lymphocytes, and eosinophils. Although all lymphocytes share the
same basic mononuclear cell morphology, lymphocyte subsets can be distinguished
by the presence (or absence) of particular proteins expressed on each cell’s
surface. The power of flow cytometry lies in its ability to detect, one cell
at a time, specific antibodies that recognize and bind to known cell surface
proteins. These antibodies serve to classify morphologically-identical lymphocytes
into their immunologically-distinct subsets.
The antibody reagents that make flow cytometric analysis possible
are known as monoclonal antibodies. A monoclonal antibody preparation is a homogenous
solution of identical antibody molecules, all of which recognize and bind to
the same, known cell surface antigen (1). A standard nomenclature has been developed
for monoclonal antibodies, as reviewed briefly in the first part of this series
(2). All monoclonal antibody preparations that recognize the same cell surface
antigen are grouped into a “Cluster of Differentiation” or CD, which
corresponds to the antigen recognized. For example, all monoclonal antibodies
which recognize a TCR-associated protein that has been assigned the name “CD3,”
are referred to as “anti-CD3” or simply as “CD3 antibodies.”
As of December 2004, CD designations had been assigned up to CD339 (4). The
CD antigens typically assessed in lymphocyte subset analyses are CD3 (all T
cells), CD3+CD4 (TH cells), CD3+CD8 (CTL-P, CTL), and CD19 or 20 (B cells).
In some cases, CD14 (monocytes) and CD16+CD56 (NK cells) would also be included
as part of an immune system assessment.
Monoclonal antibodies used in flow cytometry must be labeled so
that they can be detected if they are bound to the surface of a cell. As will
be described below, flow cytometry instruments utilize laser light for cell
analyses (Figure 2). Therefore, a monoclonal antibody reagent is labeled with
a fluorescent tag which can be detected after exposure to a particular wavelength
of light. The two most common tags which are frequently used simultaneously
are phycoerythrin (PE) and fluorescein isothiocyanate (FITC), which fluoresce
red and green, respectively. Additional tags are sometimes used, including peridinin
chlorophyll protein (PerCP) and Texas Red (3).
Prior to analysis in a flow cytometer, aliquots of whole blood
or purified WBC are incubated with labeled monoclonal antibodies, to allow binding
of the antibodies to the appropriate cell surface antigen (if present). It is
these “stained” cells that are then subjected to flow cytometric
analysis. In the schematic illustration of a flow cytometer shown in Figure 2, the WBCs being analyzed were stained simultaneously with two different monoclonal
antibodies, each recognizing a different cell surface antigen. One of the monoclonals
was tagged with a FITC label, and the other with a PE label. This has resulted
in a mixed population of cells in solution, which are shown entering the flow
cytometer at the top of the figure. This population includes cells that are
not stained by either monoclonal (i.e., do not have either antigen on their
surface), cells stained with FITC only or PE only (have only one antigen), and
cells stained with both FITC and PE (have both antigens, or “double-positive”).
The task for the flow cytometer is to analyze and record cell characteristics
that will allow enumeration of WBC and/or lymphocyte subsets based on the expression
(or lack of expression) of the cell surface antigens recognized by the monoclonal
antibodies used for staining.
Similar to hematology analyzers, flow cytometers perform their
analyses on thousands of individual cells which pass through the instrument
one at a time. As shown in Figure 2, cells are carried through the cytometer
in a focused fluid stream barely wide enough for a single cell, so that they
will pass in front of a light source one cell at a time. Virtually all flow
cytometers currently use a laser as the light source, which produces high intensity
light at a specific wavelength (3). As each cell passes in front of the laser,
it disrupts the laser beam. Each disruption is detected by several different
types of light detectors, strategically placed around the fluid stream. These
detectors make possible the multi-parameter measurements that give the flow
cytometer its analytic power.
First and foremost, the passage of a cell through the path of the laser beam
registers the presence of a cell and is the basis of the cell count. Secondly,
the manner in which the laser light is scattered provides information regarding
the physical parameters of each cell. The forward light scatter (FSC) and side
light scatter (SSC) are a function of the size and intracellular complexity
of the cell passing in front of the beam, respectively (3). These two parameters,
especially the side scatter, can be used to distinguish between granulocytes,
monocytes or macrophages, and lymphocytes. Finally, the laser light will excite
the fluorescent tag(s) (such as FITC and PE in this example) on any labeled
monoclonal antibody bound to the surface of the cell. This is illustrated in
Figure 2, where the double-positive cell currently in the laser light path is
emitting both red and green fluorescence, which are detected by their respective
detectors. When a flow cytometer is set up to detect two colors simultaneously
as shown in this example, the analysis is sometimes referred to as “five
parameter” – cell count, forward scatter, side scatter, and two
colors (usually red and green). As each of the following cells passes through
the path of the laser, they will be evaluated in the same manner. All of the
information collected on each cell is recorded and stored electronically, then
analyzed to provide the desired data regarding the entire cell population.
As an extension of the type of analysis shown in Figure 2, some
flow cytometers are capable of physically sorting cells according to their fluorescence.
This procedure is called “fluorescence-activated cell sorting” or
FACS®, hence flow cytometry analyses were sometimes referred to as “FACS
analyses.” However, since FACS® is actually a registered trademark
of Becton-Dickinson, its use as a generic term for flow cytometric analysis,
regardless of the instrument used, has been discouraged recently. This sorting
technology can be extremely useful for separating cells according to cell surface
markers, for further analysis or experimental use. However, it is not necessary
in the majority of flow cytometry analyses where an enumeration of cell types
is all that is desired.
Analysis by flow cytometry determines the relative percentages
of each cell type within the population of interest. For example, an analysis
could be performed that collected information on 5,000 lymphocytes that had
been stained with a PE-labeled antibody for CD3, an antigen which is present
on all T cells and only on T cells, and a FITC-labeled antibody for CD19, an
antigen that identifies B cells. Out of those 5,000 lymphocytes, 81% were detected
as CD3-positive (red only), 12% were CD19-positive (green only), and 7% were
found to be unstained (negative for CD3 and CD19). Therefore, of the lymphocytes,
81% are T cells, 12% are B cells, and the remainder are “non-T, non-B”
lymphocytes (usually a class of cells known as “natural killer cells”).
As another example, lymphocytes could be stained for CD3 (to identify T cells)
and CD4 (to identify the TH subset of T cells), and the percentage
of cells that were double-positive would indicate the percentage of lymphocytes
that were of the TH type.
Flow cytometry laboratories can differ widely in their reporting
format, but most do include the percentages actually obtained by the flow cytometer.
However, clinicians are often more familiar with absolute cell numbers, i.e.,
the numbers of B cells or TH cells per mm3 of blood. Therefore, even
the sophisticated flow cytometer must, in most cases, still rely on a basic
complete blood count, which would provide an absolute lymphocyte count, in order
to calculate lymphocyte subsets in absolute numbers (3). For instance, an individual
with 50% of lymphocytes registering as CD3+/CD4+ TH cells by flow
cytometric analysis, and an absolute lymphocyte count of 1800 cells/mm3 in a
hematology analysis, would have an absolute TH cell count of 900
cells/mm3. An example of reference ranges for both percentages and absolute
numbers of lymphocyte subsets can be found in the first part of this series
(2).
What is to be gained by flow cytometric analysis over a complete
blood count? It is the ability to determine in relative percentages, and calculate
in absolute numbers, the different types of lymphocytes that are critical for
different immune cell functions. While this level of discrimination is not required
for the majority of patients, it can be critical in the diagnosis and care of
persons whose medical conditions are directly related to the status of their
immune system (3). If a person has been found to have below-normal levels of
circulating immunoglobulin, it may be essential to determine if he or she has
a normal number and/or percentage of B cells. If an abnormally high percentage
or number of lymphocytes has been seen and a hematologic malignancy is suspected,
lymphocyte subset analysis specifically designed for detection of leukemia or
lymphoma could be invaluable in confirming or ruling out such a condition. In
the continuing era of the HIV/AIDS pandemic, lymphocyte subset analysis enumerating
CD4-positive TH cells, the cells which are lost as a result of HIV
infection, is considered part of routine care for HIV-infected persons.
Functional assessment of B cells as Ig-producing cells is relatively
straightfoward, since Ig is secreted by plasma cells into the surrounding lymph
fluid and the blood. Therefore, clinically-relevant information can be obtained
regarding B cell function simply by measuring the levels or types of Ig present
in the serum or plasma of peripheral blood samples (3). Such measurements may
be performed using sophisticated automated clinical laboratory instruments.
However, quantitative and qualitative determination of Ig levels, as well as
levels of many other immune system proteins (such as cytokines) and other biological
molecules of interest, can be performed utilizing relatively simple Enzyme-Linked
ImmunoSorbent Assay (ELISA) testing. Interestingly, ELISA testing can be used
to assess the immune system, while at the same time, it is also an example of
a procedure that uses immunological techniques. It harnesses the ability of
antibodies to bind with great specificity to an unprocessed antigen of interest,
without any need for APCs, enabling the detection and/or quantiation of either
soluble molecules (usually proteins) or antigen-specific antibodies in biological
fluids (3).
A schematic illustration of the most common type of ELISA, known
as an antibody capture or sandwich assay (3), is shown is Figure 3. This type
of assay is designed to detect and quantitate molecules of interest that are
soluble in serum, plasma, or any other fluid solution. The technique is called
“immunosorbent” because antibodies of known antigen-specificity,
usually monoclonal antibodies like those described above, are adsorbed or adhered
to a plastic surface. Many ELISAs are set up in plastic 96-well microwell plates,
with monoclonal antibodies coating the bottom of each well; a schematic of one
such well is shown in Figure 3. There are variations, however, that include
antibodies coated onto latex beads or membrane surfaces rather than fixed to
the bottom of a 96-well plate. A solution or liquid biological sample containing
an assortment of molecules, including the molecule of interest (represented
in Figure 3 by the indicated five-sided shape), is added to a well that has
been coated with an antibody specific for that particular molecule of interest
(top of Figure 3). After an incubation to ensure antigen-antibody binding, the
original sample solution and all other molecules are washed away, leaving only
the molecule of interest bound in the well by the monoclonal antibodies (middle
of Figure 3). A second antibody, which is specific for the same molecule but
is not blocked by the binding of antigen to the first (coating) antibody, is
added to the assay well. This second antibody binds to the antigen retained
in the well by the coating antibodies, forming a sandwich (antigen on the inside,
two different antibodies on the outside) (bottom of Figure 3). The second antibody
is known as the detection antibody, because it is labeled with an enzyme molecule
that will catalyze a color-development reaction that can be easily detected
and quantitated. It is the labeling of the detection antibody by chemically
linking or coupling an enzyme molecule to it that gives rise to the “enzyme-linked”
portion of the assay name.
Detection in an ELISA is accomplished by adding to the assay well
the appropriate liquid substrate for the enzyme label on the detection antibody.
Upon interaction with the enzyme label retained in the antigen/antibody sandwich,
the substrate changes color, and the intensity of the color is directly proportional
to the starting concentration of the molecule of interest. The color intensity
or optical density is easily determined by spectrophotometric instruments that
are specific for the appropriate wavelength of light. There are a number of
ELISA instruments available that are capable of detection in the standard 96-well
plate format as well as other formats, and accompanying computer-based software
permits both qualitative (i.e., positive or negative for the presence of the
molecule of interest) and quantitative analyses (based on comparison to standards
of known concentration).
Historically, ELISAs were preceded by a very similar technique,
known as radioimmunoassay (RIA), which utilized a radioactive isotope label
instead of an enzyme label for detection (1, 3). RIAs were originally more sensitive
than ELISAs, but with improvements in sensitivity and the appeal of the elimination
of the use of radioactive materials, ELISAs have, for the most part, replaced
RIAs as the technique of choice for determinations of this kind. This valuable
antibody-based technology, however, continues to evolve, with enzyme labels
being replaced by chemiluminescent or fluorescent labels, and fixed, single-analyte
assays in 96-well plate formats being adapted to bead-based, multiple-analyte
assays performed in solution utilizing flow cytometry for detection.
Depending on the monoclonal antibodies utilized, sandwich ELISAs,
as illustrated in Figure 3, can be designed to detect total serum Ig, or serum
levels of the subclasses of Ig (IgM, IgG, IgA, IgE). In a slightly different
type of ELISA, known as an indirect assay, a specific antigen and/or pathogen
is coated to a plate, serum or plasma is added, and the ELISA determines the
presence/absence and/or titer of antigen- or pathogen-specific antibodies in
the blood. Both of these types of analyses can shed light on the status of humoral
immunity in an individual. Further functional analysis of B cells at the cellular
level is usually beyond the scope of clinical laboratory practice, but may be
part of clinical trials or other research protocols. The techniques utilized
may include specialized flow cytometry analyses or a technique that has combined
ELISA technology with cell culture which is known as an ELISPOT assay (1, 3).
Laboratory assessment of CTL function poses a much greater challenge,
both immunologically and technically. Since CTLs must have antigens processed
and presented to them in association with self MHC molecules, it is a considerable
challenge to provide a “self” target cell. In order to prepare true
self target cells for CTL assessments, an individual would typically need to
be available to provide fresh blood samples on at least two occasions, or WBC
from a single blood sample must be frozen for future use. Additional technical
challenges include the consistent measurement of target cell killing. These
obstacles restrict the performance of CTL assessments almost exclusively to
research settings, where the time and expertise is available to carry out such
complicated assays. With the advent of more sophisticated flow cytometry techniques,
assessment of ability of T cells from an individual to recognize and respond
to a particular antigen has become possible using complexes of four synthetic
MHC molecules (known as MHC tetramers) matched to the MHC type of the individual
being assessed, and synthesized peptides that mimic processed antigens (1, 3).
Details regarding CTL assessments can be found in multiple chapters of reference
3.
As shown in Figure 1, an important early step in humoral and cytotoxic
adaptive immune responses is the activation and proliferation of the antigen-specific
T and/or B cells. Therefore, the extent of activation and/or proliferation of
lymphocytes can be used as a semi-quantitative indicator of the level of function
of the immune system (3).
There are a number of well-characterized cell surface molecules
that can act as markers of activation on lymphocytes (1, 3, 4). They tend to
be expressed on the cell surface at low or undetectable levels on resting cells,
but increase significantly following stimulation of cells. Monoclonal antibodies
that recognize and bind to activation antigens can be used to perform flow cytometry
analysis on B and T lymphocytes to determine the extent of cellular activation.
New markers and techniques for flow cytometry-based activation assays continue
to be introduced and evaluated (3).
There are various ways to evaluate cellular proliferation, usually
by measuring the extent of cellular division. Techniques range from determining
increases in cell numbers by counting live cells manually under a light microscope,
to new flow cytometry-based assays, where the intensity of staining of a cell
membrane dye is reduced by half during each cell division. However, the classic
means of determining the amount of cell division depends on the fact that all
cells must duplicate their DNA chromosomes before dividing. In such an assay,
a constant number of immune system cells is cultured in medium that contains
all four nucleoside building blocks needed for making new DNA: adenosine, cytosine,
thymidine, and guanine. As each activated lymphocyte prepares to divide, it
makes a new copy of its DNA by assembling these four nucleosides in the proper
order. In order to detect the presence of this newly-synthesized DNA (which
indicates cell division and proliferation), one of the nucleosides in the medium
is labeled with a tag that can be quantitated in some way. Thymidine (T) is
usually the labeled nucleoside, and the label used in the traditional assay
is tritium ([3H]), a radioactive isotope of hydrogen (3). Hence, this assay
is referred to as a tritiated thymidine ([3H]-T) DNA incorporation assay; if
performed on lymphocytes, it may be referred to as a lymphocyte proliferation
assay (LPA). Cultured cells are harvested onto a glass fiber filter, which captures
the newly-synthesized radioactively-labeled DNA. The amount of labeled DNA present
is determined by liquid scintillation counting, and the amount of radioactivity
present is directly proportional to the number of cell divisions which have
occurred. As is the case in all areas of the laboratory, it is desirable to
reduce or eliminate use of radioactive isotopes, so non-radioactive alternatives
have been developed for LPAs. For example, one non-radioactive DNA incorporation
assay utilizes bromodeoxyuridine (BrdUrd) as the labeled nucleoside, which can
be detected with a monoclonal antibody, while another assay uses a colorimetric,
ELISA-like assay that detects mitochondrial enzyme activity (3). Once again,
additional details regarding cellular proliferation assays can be found in reference
3.
Although most clinical laboratory scientists may never perform
any of the assays described above, this brief survey of immunologic techniques
and assessments has been presented in order to integrate the details of the
innate and adaptive cellular interactions necessary for the establishment and
maintenance of immunity into the scope of work performed in clinical laboratories.
As immunology continues to play a prominent role in clinical medicine, a broader
understanding of the complex, yet collaborative nature of immune responses should
assist the clinical laboratory scientist, regardless of his or her own area
of expertise, in participating in the complex and collaborative delivery of
clinical care.
REFERENCES
1. Kindt TJ, Goldsby RA, Osborne BA. 2006. Kuby Immunology, 6th Ed.
W.H. Freeman and Company, New York.
2. Breen EC. Feb. 2007. An Overview of the Immune System, Part 1: The Cells
and Cell Surface Molecules of the Immune System. CAMLT Newsline, Vol..
33 No. 1.
3. Rose NR, Hamilton RG, Detrick B, eds. 2002. Manual of Clinical Laboratory
Immunology, 6th Ed. American Society for Microbiology Press, Washington,
DC.
4. Human Cell Differentiation Molecules, http://www.hlda8.org/HLDAtoHCDM.htm
Figure 1
Figure 1: The Generation of Humoral and Cell-Mediated Immune Responses
A schematic illustration of the cellular interactions necessary to generate
adaptive, antigen-specific immune responses. The antigens shown are viral
particles (*), which can be both free-floating and in infected cells; processed
viral antigen is shown on cell surfaces in association with MHC Class I (rounded)
and/or MHC Class II (rectangular) molecules. Abbreviations are as follows:
antigen-presenting cell (APC), T helper cell (TH), interleukin-2 (IL-2), B
cell (B), immunoglobulin (Ig), CD8-positive cytotoxic T cell precursor (CTL-P),
cytotoxic T lymphocyte (CTL)
Figure 2: Cellular Analysis by Two-Color Flow Cytometry
A schematic representation of a laser-based flow cytometer analysis of cells
stained with fluorescent-tagged monoclonal antibodies that recognize two different
cell surface antigens. Phycoerythrin (PE)-stained cells are shown with horizontal
hatching; fluorescein isothiocyanate (FITC)-stained cells are shown with vertical
hatching; cells which stain with both antibodies (“double positive”)
are cross-hatched. Other abbreviations: side light scatter (SSC), forward
light scatter (FSC)
Figure 3
Figure 3: Sandwich Enyme-Linked ImmunoSorbent Assay (ELISA)
A schematic illustration of the steps in an ELISA designed to detect and quantitate
a specific soluble molecule of interest (five-sided shape) in a biological
fluid sample.
Review Questions Course #: DL-981 - Select
the one best answer for each question
- Observations of immunity were first recorded:
a. during an epidemic in 430 BC
b. as part of the first description of smallpox vaccination
c. by Louis Pasteur following his experiments with anthrax
d. among the survivors of the influenza epidemic of the early 1900s
- Antigen-specific immune responses are possible due to:
a. the presence of molecules on the surface of lymphocytes that act as antigen receptors
b. antigen-specific recognition of pathogens by monocytes and macrophages
c. the phagocytosis of antigen by neutrophils
d. the ability of the skin to act as a barrier to antigen
- Which of the following is not true about immune system genetic
recombination?
a. it is mediated by enzymes that physically cut and paste DNA
b. it is restricted solely to T and B cells
c. it was first described for TCR genes
d. it contributes to both specificity and diversity
- Secondary immune responses:
a. permit the invading pathogen to win the race and cause disease
b. are facilitated by a pool of antigen-specific memory B and T cells
c. are slower to occur than the primary response to the same antigen
d. occur more rapidly, but are not as vigorous as the primary response
- Cell surface immunoglobulin molecules serve as:
a. toll-like receptors on monocytes
b. antigen receptors on T cells
c. pathogen detection receptors on natural killer cells
d. antigen receptors on B cells
- Which one of the four distinguishing characteristics of
antigen-specific responses utilizes MHC molecules?
a. antigenic specificity
b. diversity
c. self/non-self recognition
d. immunologic memory
- The adaptive immune system is able to specifically rid
the body of infected or foreign cells by:
a. utilizing CTLs to induce target cell apoptosis
b. neutralizing the offending cells with antibodies
c. reducing MHC Class I expression on potential target cells
d. enhancing phagocytosis by dendritic cells
- The humoral or antibody response has evolved to deal with:
a. tumors
b. cell-associated antigens
c. tissue transplantation
d. extracellular antigens
- The adaptive immune cell type which is necessary for the
initiation of both humoral and cytotoxic immune responses is:
a. a CD4-positive T cell
b. a CD8-positive T cell
c. a CD19-positive B cell
d. an antigen-presenting cell
- The major difference in the recognition of antigen by T
cells vs. B cells is:
a. T cells recognize antigen in its native form; B cells recognize processed antigen
b. T cells recognize processed antigen plus MHC molecules; B cells recognize antigen only
c. B cells must have antigen presented to them by APC
d. B cells can recognize only protein antigens
- The ability of the adaptive immune system to specifically
recognize millions of unique sites on antigens describes:
a. antigenic specificity
b. diversity
c. self/non-self recognition
d. immunologic memory
- Cytokines are:
a. always retained within the cytoplasm of a cell until its death
b. toxic proteins released by CTLs
c. secreted proteins that send signals or messages between cells
d. markers of self and non-self
- The maturation of an antigen-activated B cell into a plasma
cell is know as:
a. apoptosis
b. proliferation
c. differentiation
d. redistribution
- Which of the following is not a property of interleukin-2
(IL-2)?
a. drives the proliferation of TH cells
b. only cytokine necessary for the differentiation of B cells
c. stimulated the division of CTL-P
d. produced by antigen-activated TH cells
- A plasma cell secretes antibody that:
a. will bind to virtually any soluble antigen encountered in the circulation
b. has the same antigen specificity as the Ig originally on the cell’s surface
c. can only deal with cell-associated antigens
d. remains sequestered in the lymph node in which it was produced
- The characteristic of antigen-specific immune responses
that is the basis for vaccination is:
a. antigenic specificity
b. diversity
c. self/non-self recognition
d. immunologic memory
- An antigen-activated CTL-P proliferates in order to:
a. begin secreting antigen-specific antibodies
b. to provide help by secreting cytokines
c. increase the number of antigen-presenting cells with the appropriate MHC expression
d. increase the number of potential CTLs with the desired antigen specificity
- A fully mature CTL can kill:
a. virtually any cell in the body expressing the appropriate antigen plus MHC Class I
b. virtually any APC presenting the appropriate antigen plus MHC Class II
c. only non-self cells expressing foreign MHC
d. only other CTLs expressing MHC Class I
- In order for TH cells to provide help to other cells, they
must do all of the following except:
a. express MHC molecules
b. provide co-stimulatory signals
c. secrete cytokines
d. recognize processed antigen
- Delayed-type hypersensitivity testing with commonly-encountered
antigens:
a. is not possible in persons who have a positive TB skin test
b. provides a quantitative measure of memory cells present for a given antigen
c. is useful as a qualitative indicator of immune system function
d. is always positive, regardless of immune system status
- Flow cytometric analysis:
a. is useful only when standard hematology analysis is unavailable
b. utilizes a spectrophotometric instrument
c. is unable to distinguish between morphologically-identical B and T cells
d. permits enumeration of lymphocyte subsets
- For use in flow cytometry analyses, a monoclonal antibody
must:
a. be inherently reactive to laser light
b. recognize several different antigens on a single cell
c. be labeled so that it can be detected when bound to cells
d. reflect only white light
- Of the four distinguishing characteristics of adaptive
immune responses, which one enables the immune system to discriminate between
proteins that differ only by a single amino acid?
a. antigenic specificity
b. diversity
c. self/non-self recognition
d. immunologic memory
- Which of the following is not a parameter measured by a
flow cytometer as a cell passes through its laser beam?
a. electrical resistance
b. forward scattered light
c. side scattered light
d. presence/absence of fluorescence
- The status of an individual’s humoral immunity is easily
assessed by:
a. enumeration of CD3+ cells by flow cytometry
b. determination of serum levels of Ig
c. induction of apoptosis in target cells
d. measuring secretion of IL-2
- Plate-based ELISA technology utilizes:
a. MHC-matched self target cells
b. a radioactive isotope as its label
c. a laser-based instrument
d. enzyme-linked antigen-specific detection antibodies
- Which of the following would not be necessary in order
to assess CTL function?
a. self target cell
b. MHC class I molecules
c. CD8 T cells
d. soluble antigen
- The classic techniques for measurement of lymphocyte proliferation
depend upon:
a. the generation of non-dividing memory T and B cells
b. the processing and presentation of self DNA by APCs
c. the synthesis of new DNA prior to cell division
d. the incorporation of radioactive thymidine into cell membranes
- All of the following are alternative techniques for measuring
lymphocyte proliferation or activation except:
a. flow cytometry-based assay utilizing a cell membrane dye that is reduced by half during each cell division
b. flow cytometry analysis utilizing monoclonal antibodies that recognize activation antigens
c. indirect ELISA utilizing cellular DNA to determine the titer of anti-DNA antibodies
d. non-radioactive DNA incorporation assay utilizing bromodeoxyuridine
- The generation of immune responses:
a. has no bearing on the delivery of clinical care
b. has little or no impact on the practice of medicine today
c. can only ever be assessed by research scientists
d. is a collaborative effort of the adaptive and innate immune systems