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✪ B-lymphocytes and T-lymphocytes of CD4+ and CD8+ populations

✪ Cytotoxic T-lymphocytes

✪ T-lymphocytes

✪ Lymphocytes

✪ B-lymphocytes (B-cells)

Subtitles

I have already talked about the main cells of a specific immune system and now we recap what we have learned. Let's start with the B-lymphocyte, which I always draw in blue.. Here it is in front of you. Membrane immunoglobulins are present on the surface of B-lymphocytes, and each such lymphocyte has its own variant of the variable domain. I repeat: B-lymphocytes have membrane immunoglobulins on the surface, and each such lymphocyte has its own version of the variable domain. I'll draw the variable domains in pink. Another B-lymphocyte will have different variable domains. Therefore, they can respond to a variety of antigens that have entered the body. In this case, B-lymphocytes are activated. What is needed for this and what happens in this case? Let's talk about what happens when B-lymphocytes are activated. What do you need to start activation? This requires the pathogen to bind to the membrane immunoglobulin. We write that the pathogen binds. The pathogen binds to membrane immunoglobulin. But this is not enough. Normally, a B-lymphocyte needs stimulation from a T-lymphocyte. So we write: stimulation by a T-lymphocyte. In what situation is such stimulation necessary? B-lymphocyte is an antigen-presenting cell. It absorbs the antigen, cleaves it and shows it along with MHC class 2. We will also draw it now. This is class 2 MHC. Antigen fragments bind to it. This complex binds to an activated T helper, which has a receptor with a variable domain specific for that particular antigen. Yes, the receptor turned out to be crooked, but the essence is clear, at least I will hope so. After activation, differentiation follows: the cell divides, and its descendants can become effector cells. This is true for both T- and B-lymphocytes. Once activated, the lymphocyte produces effector and memory cells. Memory cells are stored for a long time, and as a result of division, a lot of them are obtained. When the same pathogen re-enters, it is more likely to stumble upon the memory cell, triggering a rapid immune response. Effector B-lymphocytes are factories for the production of immunoglobulins. So, effector B-lymphocytes - produce immunoglobulin. The logic is this: since the antibody approaches the antigen that has entered the body, more should be synthesized. All the production capacity of the cell is taken to synthesize antibodies. I'll tell you one fact that my wife suggested to me. Overhearing how I recorded the last video. She is a specialist in hematology and understands immunology, so I trust her in this: she is an expert in this matter. In the last video, I recklessly stated that antibodies are produced by activated effector B-lymphocytes. So it really is - antibodies are produced exclusively by B-lymphocytes. However, antibody-secreting cells have their own name. These effector B lymphocytes are commonly referred to as plasma cells. I'll write down the term. In the course of differentiation, the name changes. This is the name of the B-lymphocyte, which began to secrete antibodies. Thereafter, it is exclusively referred to as a plasma cell. So when asked which cells produce antibodies, do not answer that they are B-lymphocytes. The correct answer is: plasma cells. This is a common term used in immunology as well as rheumatology. Excuse me, did I say my wife is a hematologist? No, she's a rheumatologist. Sometimes I get confused about this. So, the essence of B-lymphocytes is the production of antibodies that will bind to the antigens of viruses or bacteria and make them visible to macrophages and other phagocytes. But that's all about them, now let's move on to T-lymphocytes. I will tell about them what was not in the previous videos. So, there are two types of T-lymphocytes. You already know about helpers and cytotoxic T-lymphocytes, but there is another classification of lymphocytes, and I will tell you about it. So there are two varieties. Both have a T-cell receptor. I'll draw it like this. T-cell receptor. In addition, there are a number of other proteins on their membranes. Some T-lymphocytes have a membrane protein called CD4. CD4. Other T-lymphocytes have another protein - this is CD8. We'll sign it too. CD8. The lymphocyte on the right is called a CD8-positive T-lymphocyte. It has CD8 on its membrane. And here is a CD4-positive T-lymphocyte. Here are two varieties. They are divided according to these proteins. The CD4 protein is a receptor that has an affinity for MHC class 2 proteins. Most CD4-positive cells are T helpers. In most cases, if CD4-positive cells are mentioned in a conversation, then out of habit they mean precisely helper T-lymphocytes. They usually talk about them. Perhaps I'll sign it - T-helper. The CD8 receptor has an affinity for MHC class 1. We indicate this in the figure. In cancer cells, MHC class 1 on the membrane is associated with cancer antigens. Therefore, CD8 is characteristic of cytotoxic lymphocytes. CD8 is characteristic of cytotoxic lymphocytes. Usually, before the cell is activated, it is called CD4- or CD8-positive, and the function of the lymphocyte is said after activation. Already after. These are terminological features. I hope you get the gist. Now let's remember what this lymphocyte does. It binds to MHC proteins that are found on the membrane along with antigens. Here is class 1 MHC. As I said in the last video, every cell with a nucleus has it. Let's say something bad happened in the cell. Something bad, maybe it's a virus. Maybe cancer. The affected cell must die, otherwise it will copy the virus or multiply if it is a tumor. So, CD8-positive T-lymphocytes kill cells affected by a virus or oncology. They kill affected cells that might otherwise threaten the entire body as a whole. T-helpers are a completely different matter. Let's take dendritic cell an antigen-presenting cell. She has MHC class 2, to which fragments of the digested antigen are connected. It activates helper T-lymphocytes, which divide and differentiate into effector and memory cells. The effector T-lymphocyte has several functions. Helper T-lymphocyte activates B-lymphocytes and releases cytokines. Releases cytokines. An activated lymphocyte releases many substances that serve as a signal to other cells, such as other lymphocytes, while raising the alarm. Some of these cytokines help cytotoxic lymphocytes in their activation. Cytokines raise the alarm and CD8-positive, that is, cytotoxic T-lymphocytes, effector lymphocytes, are taken to kill the cells. As for memory cells, these are copies of the original lymphocytes that are permanently stored in this place in case of a repeat of the threat, in order to provide a faster response. I hope that I didn’t confuse you too much with new terms, but it was necessary. And now you know that antibodies are synthesized not by B-lymphocytes, not by them, but by cells that have their own name. These are plasma cells or plasmocytes.

Types of T-lymphocytes

T-lymphocytes that provide central regulation of the immune response.

Differentiation in the thymus

All T cells originate from hematopoietic red bone marrow stem cells that migrate to the thymus and differentiate into immature thymocytes. The thymus creates the microenvironment necessary for the development of a fully functional T cell repertoire that is MHC-limited and self-tolerant.

Thymocyte differentiation is divided into different stages depending on the expression of various surface markers (antigens). On the very early stage, thymocytes do not express CD4 and CD8 co-receptors and are therefore classified as double negative (English Double Negative (DN)) (CD4-CD8-). At the next stage, thymocytes express both coreceptors and are called double positive (Eng. Double Positive (DP) ) (CD4+CD8+). Finally, at the final stage, cells are selected that express only one of the co-receptors (eng. Single Positive (SP)): either (CD4+) or (CD8+).

The early stage can be divided into several sub-stages. So, at the DN1 substage (English Double Negative 1 ), thymocytes have the following combination of markers: CD44 + CD25 -CD117 +. Cells with this combination of markers are also called early lymphoid progenitors. Early Lymphoid Progenitors (ELP)). Progressing in their differentiation, ELP actively divide and finally lose the ability to transform into other cell types (for example, B-lymphocytes or myeloid cells). Going to the DN2 substage (eng. Double Negative 2 ), thymocytes express CD44 + CD25 + CD117 + and become early T-cell progenitors (eng. Early T-cell Progenitors (ETP)). During the DN3 substage (eng. Double Negative 3 ), ETP cells have a combination of CD44-CD25 + and enter into the process β-selection.

β selection

The T-cell receptor genes consist of repeating segments belonging to three classes: V (eng. variable), D (eng. diversity) and J (eng. joining). During somatic recombination, gene segments, one from each class, are linked together (V(D)J recombination). Random combination of V(D)J segment sequences results in unique variable domain sequences for each of the receptor chains. The random nature of the formation of sequences of variable domains allows the generation of T cells that can recognize a large number of different antigens, and, as a result, provide more effective protection against rapidly evolving pathogens. However, this same mechanism often leads to the formation of non-functional subunits of the T-cell receptor. The genes encoding the β-subunit of the receptor are the first to undergo recombination in DN3 cells. To exclude the possibility of the formation of a non-functional peptide, the β-subunit forms a complex with the invariable α-subunit of the pre-T-cell receptor, forming the so-called. pre-T cell receptor (pre-TCR). Cells unable to form functional pre-TCR die by apoptosis. Thymocytes that have successfully passed β-selection move to the DN4 substage (CD44 -CD25 -) and undergo the process positive selection.

positive selection

Cells that express pre-TCR on their surface are still not immunocompetent, since they are not able to bind to molecules of the major histocompatibility complex. Recognition of MHC molecules by the T-cell receptor requires the presence of CD4 and CD8 co-receptors on the surface of thymocytes. The formation of a complex between pre-TCR and the CD3 coreceptor leads to inhibition of rearrangements of the β-subunit genes and, at the same time, causes activation of the expression of the CD4 and CD8 genes. Thus thymocytes become double positive (DP) (CD4+CD8+). DP-thymocytes actively migrate to the thymus cortex, where they interact with cortical epithelial cells expressing proteins of both classes of MHC (MHC-I and MHC-II). Cells that are unable to interact with MHC proteins of the cortical epithelium undergo apoptosis, while cells that successfully carry out such an interaction begin to actively divide.

negative selection

Thymocytes that have undergone positive selection begin to migrate to the cortico-medullary border of the thymus. Once in the medulla, thymocytes interact with the body's own antigens, presented in combination with MHC proteins on medullary thymic epithelial cells (mTECs). Thymocytes actively interacting with their own antigens undergo apoptosis. Negative selection prevents the emergence of self-activating T cells capable of causing autoimmune diseases of the clone. Some of the cells in this clone turn into effector T cells, which perform functions specific to this type of lymphocyte (for example, they secrete cytokines in the case of T-helpers or lyse the affected cells in the case of T-killers). Another part of the activated cells is transformed into T-cells memory. Memory cells remain in an inactive form after initial contact with an antigen until repeated interaction with the same antigen occurs. Thus, memory T-cells store information about previously acting antigens and provide a secondary immune response that is carried out in a shorter time than the primary one.

The interaction of the T-cell receptor and co-receptors (CD4, CD8) with the major histocompatibility complex is important for the successful activation of naive T cells, but it is not sufficient by itself for differentiation into effector cells. For the subsequent proliferation of activated cells, the interaction of the so-called. costimulatory molecules. For T helpers, these molecules are the CD28 receptor on the surface of the T cell and immunoglobulin B7 on the surface of the antigen presenting cell.

HIV is a virus that attacks the immune system. In our immune system, there are a large number of cells that perform various functions:

• Leukocytes;
• Phagocytes;
• macrophages;
• Neutrophils;
• T-helpers (CD4-lymphocytes);
• T-killers.

Each of these cells is responsible for a certain stage of the response to a foreign object. HIV infects only one group of cells - CD4 lymphocytes (T-lymphocytes). They are responsible for recognizing a foreign gene.

By the number of certain cells, the doctor draws conclusions about the patient's condition. The AIDS test is based on the number of T-lymphocytes (CD4-lymphocytes) in a blood sample.

Diseases for which a doctor may order an AIDS test

If the blood test shows unspecified diseases connective tissue, an inflammatory process, an HIV test may be prescribed. A good marker of HIV is a sharp decrease in CD4-lymphocytes. In the case when other infections and a predisposition to a certain group of diseases (colds, for example) are detected, an HIV test is not performed.

Important! If an inflammatory process that has no basis is found, it is necessary to take an HIV test.

Do not be afraid if the doctor starts talking about an HIV test. The diagnosis may not be confirmed. With a positive result, it is important to start treatment as soon as possible.

Norms

• Overwork of the body;
• Menstrual cycle;
• epidemiological environment;
• Some medicines.

The number of T-lymphocytes (helpers) is restored after rest.

If the absolute CD4 count does not recover within a certain period, the doctor may order an HIV test.

Interpretation of the result of the analysis for AIDS

At healthy person all indicators should be normal. If one of the parameters is changed, a viral load test is assigned. After that, the results of the blood test are correlated with this indicator. This will help you determine the cause of the violation.

The lymphocyte count decreases in the case of an infectious disease, but is restored after a course of treatment up to normal level. There will be no improvement in system performance in HIV patients. The test is based on this.

What is immune status

When determining the immune status of a person, blood parameters are examined:

• Total and relative number of lymphocytes;
• Number of t-lymphocyte helpers;
• Phagocytic activity of macrophages;
• Changes in immunoglobulins of different classes.

Of all the above, only T-lymphocytes are specific to HIV.

Important! A decrease in CD4-lymphocytes indicates a terrible disease. An increase in their level indicates another inflammatory process.

What does the CD4 count say?

CD4 cells are contained in the blood in a certain amount. If there is a decrease in them, the body quickly restores the number. When the immune system is suppressed, the number of lymphocytes decreases, the activity of T-suppressors, on the contrary, leads to the activation of protective forces.

Viral cells multiply very quickly, so when infected with HIV, the level of T-lymphocytes cannot recover to normal levels.

Changes in CD4 count

CD4 cells are the first to respond to the penetration of a foreign agent into the body. A decrease in the level indicates a high activity of the virus.

The number of cells/µl may vary depending on:

• Time of day (in the morning it is higher);
• The presence of infectious diseases;
• The process of processing blood (with the wrong procedure, cells can be destroyed);
• The medications taken (hormonal and steroid drugs significantly affect this indicator).

Percentage of CD4

When testing for HIV, blood counts are often expressed as a percentage.

Helpers CD3, D8, CD19, CD16+56, as well as the ratio of CD4 CD8 decreases with a decrease in the immune status. But these parameters do not indicate HIV.

Only the CD4 helper is specific to the immunodeficiency virus:

• If its content is 12-15%, then in terms of the blood contains 200 cells / mm 3;
• At values ​​from 29%, the content of cells is from 450 cells/mm 3 ;

In an HIV-negative person, the value of this parameter is 40%.

When immune cells are damaged, immunity decreases. to determine the rate of this process, the viral load is calculated - the amount of foreign RNA per ml of blood. This parameter is predictive.

The immune system of women is weaker, so the viral load indicator, according to the results of the study, begins to decline much earlier than in men.

What does an undetectable viral load mean?

The viral load indicator may not be determined after a few months. Depending on the activity of the virus, its number in the blood may vary. Then, with a low sensitivity of the apparatus, it will not detect the virus.

Important! An indeterminate viral load does not mean that the virus has completely disappeared. Treatment for AIDS should not be stopped, because without treatment, remission will occur and the amount of the virus will increase.

The effect of vaccinations and infections

Vaccination or an infectious disease temporarily increases the viral load. Taking prophylactic drugs, on the contrary, reduces. To accurately determine the immune status after the above procedures, you should wait for some time. The period will be set by the doctor depending on the circumstances.

What are the benefits of an undetectable viral load?

In HIV-positive people, an undetectable viral load can occur if:

• Proper antiretroviral therapy;
• Low progression of the virus.

This contributes to the normalization of the patient's condition. With multiple repeated courses, immunological tolerance may develop. The immunological response in this case ceases to respond to treatment. In this case, it is necessary to change the course of treatment. This can happen if:

• The course of treatment was not completed;
• The same course was repeated several times in a row;
• Individual insensitivity to prescribed drugs.

natural variations

The virus can be in the body in several stages:

• incubation stage;
• Period of acute infection;
• latent stage;
• Stage of secondary diseases;

During different periods of activity, viral load indicators change significantly. Within a few days, this parameter can change three times, regardless of the course of treatment. Sharp short-term jumps may not affect the health of the patient. The determination of drug resistance is carried out several times. The final result is calculated as an average.

The use of suppressors leads to the stabilization of the number of viruses in the blood.

Significant changes

If the number of HIV viruses remains high for several months, it is worth paying attention to this. Important indicators exceeding the norm by 3-5 times. If the increase in the level of CD4-lymphocytes passes during the course of treatment, it may be necessary to change medications, since the body has lost its sensitivity to them.

Variance minimization

When taking an analysis for the amount of immunodeficiency virus, CD4-lymphocytes in the blood, it should be understood that different devices have different sensitivities. It may differ depending on the instrument brand or calibration value. In order to minimize the error associated with the devices, the analysis should be taken in the same clinic on the same device.

If one of the partners in the family is HIV-positive, there is a certain schedule in sexual life. If the viral load rises, you should completely refrain from sexual contact, as the likelihood of infection increases significantly.

By lowering the threshold for the number of viruses, using certain medications on the recommendation of a doctor, sexual activity can be resumed.

What is the threshold for determining current tests

Sensitive modern tests for the diagnosis of HIV is gradually increasing. Most devices in Russia are sensitive to the amount of virus 400-500 pieces/ml of blood. Some more expensive devices detect the virus by the standard method at a count of 50/ml.

The literature indicates that some modern models are able to recognize HIV at a population of only 2 pieces/ml of blood, but such technologies are not yet used in hospitals and private clinics.

Mistakes

Despite the high sensitivity of modern devices, errors still occur in determining viral load values. They are associated with:

• Incorrect calibration of the device;
• Poor handling of flasks from previous assays;
• Incorrectly prepared blood sample;
• Presence in the blood medicines reducing sensitivity.

These errors are corrected by re-analyzing the same blood sample or a new portion.

Decision to start antiretroviral therapy

If tests show high value viral load over a long period of time, the doctor decides on the appointment of a course of treatment. Start of treatment HIV infection and taking medications does not begin immediately, but gradually. Most drugs are introduced into the course of treatment over a certain period, so that the body gets used to a significant amount of aggressive chemical components. The number of CD4 lymphocytes in the blood plays an important role in making such a decision.

In the event that a person cannot or does not want to start treatment, he must constantly take an analysis and monitor the level of lymphocytes in the blood.

Advice! If you have not started antiretroviral therapy, get tested for HIV and your CD4 count on a regular basis. If you miss a critical minimum, the body may not be able to cope. Recovery will take much more time, money and effort.

If you have an increase in viral load while on therapy

If the viral load continues to increase after starting treatment, there may be two options:

• Not enough treatment time has passed to restore normal parameters;
• The body is not sensitive to prescribed drugs.

The decision on further actions is made by the doctor based on the tests and the patient's condition.

How to improve your viral load test results

As a result proper treatment the amount of cd4 in the blood should be gradually restored.

This will also help:

• Proper nutrition;
• Rejection of bad habits;
• No stress;
• No fatigue.

If you are not taking antiretroviral therapy

When deciding whether to start a course of treatment or not, it is important to understand what antiretroviral therapy is for HIV AIDS. These drugs are aimed at suppressing the activity of the virus outside the cells of the body. Due to this, during therapy, the immune system is restored in patients.

In the complex of drugs there are also those that contribute to the restoration of the body's natural defenses.

In the absence of such therapy, the virus has the ability to multiply freely, affecting more and more cells of the host's immune system.

agammaglobulinemia(agammaglobulinemia; a- + gamma globulins + gr. haima blood; synonym: hypogammaglobulinemia, antibody deficiency syndrome) - the general name of a group of diseases characterized by the absence or a sharp decrease in the level of immunoglobulins in the blood serum;

autoantigens(auto- + antigens) - the body's own normal antigens, as well as antigens that arise under the influence of various biological and physico-chemical factors, in relation to which autoantibodies are formed;

autoimmune reaction- the body's immune response to autoantigens;

allergy (allergies; Greek allos other, different + Ergon action) - a state of altered reactivity of the organism in the form of an increase in its sensitivity to repeated exposure to any substances or to components of its own tissues; Allergy is based on an immune response that occurs with tissue damage;

active immunity immunity resulting from the body's immune response to the introduction of an antigen;

The main cells that carry out immune reactions are T- and B-lymphocytes (and derivatives of the latter - plasma cells), macrophages, as well as a number of cells interacting with them (mast cells, eosinophils, etc.).

• Lymphocytes

The population of lymphocytes is functionally heterogeneous. There are three main types of lymphocytes: T-lymphocytes, B-lymphocytes and the so-called zero lymphocytes (0-cells). Lymphocytes develop from undifferentiated lymphoid bone marrow progenitors and, upon differentiation, acquire functional and morphological features (presence of markers, surface receptors) detected by immunological methods. 0-lymphocytes (null) are devoid of surface markers and are considered as a reserve population of undifferentiated lymphocytes.

T-lymphocytes- the most numerous population of lymphocytes, constituting 70-90% of blood lymphocytes. They differentiate in the thymus gland - thymus (hence their name), enter the blood and lymph and populate T-zones in the peripheral organs of the immune system - lymph nodes (deep part of the cortical substance), spleen (periarterial sheaths of lymphoid nodules), in single and multiple follicles of various organs, in which T-immunocytes (effector) and T-memory cells are formed under the influence of antigens. T-lymphocytes are characterized by the presence on the plasmalemma of special receptors that can specifically recognize and bind antigens. These receptors are products of immune response genes. T-lymphocytes provide cellular immunity, participate in the regulation of humoral immunity, carry out the production of cytokines under the action of antigens.

In the population of T-lymphocytes, several functional groups of cells are distinguished: cytotoxic lymphocytes (Tc), or T-killers(TK), T-helpers(Tx), T-suppressors(Ts). TK are involved in cellular immunity reactions, ensuring the destruction (lysis) of foreign cells and their own altered cells (for example, tumor cells). The receptors allow them to recognize the proteins of viruses and tumor cells on their surface. At the same time, the activation of Tc (killers) occurs under the influence of histocompatibility antigens on the surface of foreign cells.

In addition, T-lymphocytes are involved in the regulation of humoral immunity with the help of Tx and Tc. Tx stimulate the differentiation of B-lymphocytes, the formation of plasma cells from them and the production of immunoglobulins (Ig). Tx have surface receptors that bind to proteins on the plasmolemma of B cells and macrophages, stimulating Tx and macrophages to proliferate, produce interleukins (peptide hormones), and B cells to produce antibodies.

Thus, the main function of Tx is the recognition of foreign antigens (presented by macrophages), the secretion of interleukins that stimulate B-lymphocytes and other cells to participate in immune responses.

A decrease in the number of Tx in the blood leads to a weakening defensive reactions body (these individuals are more susceptible to infections). A sharp decrease in the number of Tx in persons infected with the AIDS virus was noted.

Tc are able to inhibit the activity of Tx, B-lymphocytes and plasma cells. They are involved in allergic reactions, hypersensitivity reactions. Tc suppress the differentiation of B-lymphocytes.

One of the main functions of T-lymphocytes is the production cytokines, which have a stimulating or inhibitory effect on the cells involved in the immune response (chemotactic factors, macrophage inhibitory factor - MIF, non-specific cytotoxic substances, etc.).

natural killers. Among the lymphocytes in the blood, in addition to the above-described Tc, which perform the function of killers, there are so-called natural killers (Hk, NK), which are also involved in cellular immunity. They form the first line of defense against foreign cells, act immediately, quickly destroying cells. NK in their own body destroy tumor cells and cells infected with the virus. Tc form a second line of defense, since it takes time for them to develop from inactive T-lymphocytes, so they come into action later than Hc. NK are large lymphocytes with a diameter of 12-15 microns, have a lobed nucleus and azurophilic granules (lysosomes) in the cytoplasm.

• Development of t- and b-lymphocytes

The ancestor of all cells of the immune system is the hematopoietic stem cell (HSC). HSCs are localized in the embryonic period in the yolk sac, liver, and spleen. In the later period of embryogenesis, they appear in the bone marrow and continue to proliferate in postnatal life. HSCs in the bone marrow produce a lymphopoietic progenitor cell (lymphoid multipotent progenitor cell) that generates two types of cells: pre-T cells (progenitors of T cells) and pre-B cells (progenitors of B cells).

• T-lymphocyte differentiation

Pre-T cells migrate from the bone marrow through the blood to the central organ of the immune system, the thymus gland. Even during the period of embryonic development, a microenvironment is created in the thymus gland, which is important for the differentiation of T-lymphocytes. In the formation of the microenvironment, a special role is assigned to the reticuloepithelial cells of this gland, which are capable of producing a number of biologically active substances. Pre-T cells migrating to the thymus acquire the ability to respond to microenvironmental stimuli. Pre-T cells in the thymus proliferate, transform into T-lymphocytes carrying characteristic membrane antigens (CD4+, CD8+). T-lymphocytes generate and “deliver” into the blood circulation and thymus-dependent zones of peripheral lymphoid organs of 3 types of lymphocytes: Tc, Tx and Tc. The "virgin" T-lymphocytes migrating from the thymus (virgile T-lymphocytes) are short-lived. Specific interaction with an antigen in peripheral lymphoid organs initiates the processes of their proliferation and differentiation into mature and long-lived cells (T-effector and T-memory cells), which make up the majority of recirculating T-lymphocytes.

Not all cells migrate from the thymus gland. Part of T-lymphocytes dies. There is an opinion that the cause of their death is the attachment of an antigen to an antigen-specific receptor. There are no foreign antigens in the thymus, so this mechanism can serve to remove T-lymphocytes that can react with the body's own structures, i.e. perform the function of protection against autoimmune reactions. The death of some lymphocytes is genetically programmed (apoptosis).

T cell differentiation antigens. In the process of differentiation of lymphocytes, specific membrane molecules of glycoproteins appear on their surface. Such molecules (antigens) can be detected using specific monoclonal antibodies. Received monoclonal antibodies that react with only one antigen cell membrane. Using a set of monoclonal antibodies, subpopulations of lymphocytes can be identified. There are sets of antibodies to differentiation antigens of human lymphocytes. Antibodies form relatively few groups (or "clusters"), each of which recognizes a single cell surface protein. A nomenclature of differentiation antigens of human leukocytes, detected by monoclonal antibodies, has been created. This CD nomenclature ( CD - cluster of differentiation- differentiation cluster) is based on groups of monoclonal antibodies that react with the same differentiation antigens.

Polyclonal antibodies to a number of differentiating antigens of human T-lymphocytes have been obtained. When determining the total population of T cells, monoclonal antibodies of CD specificities (CD2, CD3, CDS, CD6, CD7) can be used.

Differentiating antigens of T cells are known, which are characteristic either for certain stages of ontogeny or for subpopulations that differ in functional activity. Thus, CD1 is a marker of the early phase of T-cell maturation in the thymus. During the differentiation of thymocytes, CD4 and CD8 markers are simultaneously expressed on their surface. However, subsequently, the CD4 marker disappears from a part of the cells and remains only on the subpopulation that has ceased to express the CD8 antigen. Mature CD4+ cells are Th. The CD8 antigen is expressed on about ⅓ of peripheral T cells that mature from CD4+/CD8+ T lymphocytes. The subpopulation of CD8+ T cells includes cytotoxic and suppressor T lymphocytes. Antibodies to the CD4 and CD8 glycoproteins are widely used to distinguish and separate T cells into Tx and Tc, respectively.

In addition to differentiation antigens, specific markers of T-lymphocytes are known.

T-cell receptors for antigens are antibody-like heterodimers consisting of polypeptide α- and β-chains. Each of the chains is 280 amino acids long, and the large extracellular portion of each chain is folded into two Ig-like domains: one variable (V) and one constant (C). The antibody-like heterodimer is encoded by genes that are assembled from several gene segments during the development of T cells in the thymus.

There are antigen-independent and antigen-dependent differentiation and specialization of B- and T-lymphocytes.

Antigen-independent proliferation and differentiation are genetically programmed for the formation of cells capable of giving a specific type of immune response when they encounter a specific antigen due to the appearance of special “receptors” on the plasmolemma of lymphocytes. It takes place in the central organs of immunity (thymus, Bone marrow or bursa of Fabricius in birds) under the influence of specific factors produced by cells that form the microenvironment (reticular stroma or reticuloepithelial cells in the thymus).

antigen dependent proliferation and differentiation of T- and B-lymphocytes occur when they encounter antigens in peripheral lymphoid organs, with the formation of effector cells and memory cells (retaining information about the acting antigen).

The resulting T-lymphocytes form a pool long-lived, recirculating lymphocytes, and B-lymphocytes - short lived cells.

66. Characteristics of B-lymphocytes.

B-lymphocytes are the main cells involved in humoral immunity. In humans, they are formed from the SCM of the red bone marrow, then enter the bloodstream and then populate the B-zones of peripheral lymphoid organs - the spleen, lymph nodes, lymphoid follicles of many internal organs. Their blood contains 10-30% of the entire population of lymphocytes.

B-lymphocytes are characterized by the presence of surface immunoglobulin receptors (SIg or MIg) for antigens on the plasmalemma. Each B cell contains 50,000-150,000 antigen-specific SIg molecules. In the population of B-lymphocytes there are cells with various SIg: the majority (⅔) contain IgM, a smaller number (⅓) contain IgG, and about 1-5% contain IgA, IgD, IgE. In the plasma membrane of B-lymphocytes, there are also receptors for complement (C3) and Fc receptors.

Under the action of the antigen, B-lymphocytes in peripheral lymphoid organs are activated, proliferate, differentiate into plasma cells, actively synthesizing antibodies of various classes, which enter the blood, lymph and tissue fluid.

Differentiation of B-lymphocytes

B-cell precursors (pre-B-cells) develop further in birds in the bursa of Fabricius (bursa), whence the name B-lymphocytes came from, in humans and mammals - in the bone marrow.

Bag of Fabricius (bursa Fabricii) - the central organ of immunopoiesis in birds, where the development of B-lymphocytes occurs, is located in the cloaca. Its microscopic structure is characterized by the presence of numerous folds covered with epithelium, in which lymphoid nodules are located, bounded by a membrane. The nodules contain epitheliocytes and lymphocytes at various stages of differentiation. During embryogenesis, a brain zone is formed in the center of the follicle, and a cortical zone is formed on the periphery (outside the membrane), into which lymphocytes from the brain zone probably migrate. Due to the fact that only B-lymphocytes are formed in the bursa of Fabricius in birds, it is a convenient object for studying the structure and immunological characteristics of this type of lymphocytes. The ultramicroscopic structure of B-lymphocytes is characterized by the presence of groups of ribosomes in the form of rosettes in the cytoplasm. These cells have larger nuclei and less dense chromatin than T-lymphocytes due to the increased euchromatin content.

B-lymphocytes differ from other cell types in their ability to synthesize immunoglobulins. Mature B-lymphocytes express Ig on the cell membrane. Such membrane immunoglobulins (MIg) function as antigen-specific receptors.

Pre-B cells synthesize intracellular cytoplasmic IgM but lack surface immunoglobulin receptors. Bone marrow virgil B lymphocytes have IgM receptors on their surface. Mature B-lymphocytes carry on their surface immunoglobulin receptors of various classes - IgM, IgG, etc.

Differentiated B-lymphocytes enter the peripheral lymphoid organs, where, under the action of antigens, proliferation and further specialization of B-lymphocytes occur with the formation of plasma cells and memory B-cells (VP).

During their development, many B cells switch from producing antibodies of one class to producing antibodies of other classes. This process is called class switching. All B cells begin their antibody synthesis activity by producing IgM molecules, which are incorporated into the plasma membrane and serve as antigen receptors. Then, even before interacting with the antigen, most of the B cells proceed to the simultaneous synthesis of IgM and IgD molecules. When a virgil B cell switches from producing membrane-bound IgM alone to simultaneously producing membrane-bound IgM and IgD, the switch is likely due to a change in RNA processing.

When stimulated with an antigen, some of these cells become activated and begin to secrete IgM antibodies, which predominate in the primary humoral response.

Other antigen-stimulated cells switch to producing IgG, IgE, or IgA antibodies; Memory B cells carry these antibodies on their surface, and active B cells secrete them. IgG, IgE, and IgA molecules are collectively referred to as secondary class antibodies because they appear to be formed only after antigen challenge and predominate in secondary humoral responses.

With the help of monoclonal antibodies, it was possible to identify certain differentiation antigens, which, even before the appearance of cytoplasmic µ-chains, make it possible to attribute the lymphocyte carrying them to the B-cell line. Thus, the CD19 antigen is the earliest marker that allows one to attribute a lymphocyte to the B-cell series. It is present on pre-B cells in the bone marrow, on all peripheral B cells.

The antigen detected by monoclonal antibodies of the CD20 group is specific for B-lymphocytes and characterizes the later stages of differentiation.

On histological sections, the CD20 antigen is detected on B-cells of the germinal centers of lymphoid nodules, in the cortical substance of the lymph nodes. B-lymphocytes also carry a number of other (eg, CD24, CD37) markers.

67. Macrophages play an important role in both natural and acquired immunity of the body. The participation of macrophages in natural immunity is manifested in their ability to phagocytosis and in the synthesis of a number of active substances - digestive enzymes, components of the complement system, phagocytin, lysozyme, interferon, endogenous pyrogen, etc., which are the main factors of natural immunity. Their role in acquired immunity consists in the passive transfer of antigen to immunocompetent cells (T- and B-lymphocytes), in the induction of a specific response to antigens. Macrophages are also involved in providing immune homeostasis by controlling the reproduction of cells characterized by a number of abnormalities (tumor cells).

For the optimal development of immune responses under the action of most antigens, the participation of macrophages is necessary both in the first inductive phase of immunity, when they stimulate lymphocytes, and in its final phase (productive), when they participate in the production of antibodies and destruction of the antigen. Antigens phagocytosed by macrophages elicit a stronger immune response than those not phagocytosed by them. Blockade of macrophages by introducing a suspension of inert particles (for example, carcasses) into the body of animals significantly weakens the immune response. Macrophages are capable of phagocytizing both soluble (for example, proteins) and particulate antigens. Corpuscular antigens elicit a stronger immune response.

Some types of antigens, such as pneumococci, containing a carbohydrate component on the surface, can be phagocytized only after preliminary opsonization. Phagocytosis is greatly facilitated if the antigenic determinants of foreign cells are opsonized, i.e. linked to an antibody or an antibody-complement complex. The opsonization process is provided by the presence of receptors on the macrophage membrane that bind part of the antibody molecule (Fc fragment) or part of the complement (C3). Only antibodies of the IgG class can directly bind to the macrophage membrane in humans when they are in combination with the corresponding antigen. IgM can bind to the macrophage membrane in the presence of complement. Macrophages are able to "recognize" soluble antigens, such as hemoglobin.

In the mechanism of antigen recognition, two stages are closely related to each other. The first step is phagocytosis and digestion of the antigen. In the second stage, macrophage phagolysosomes accumulate polypeptides, soluble antigens (serum albumins), and corpuscular bacterial antigens. Several introduced antigens can be found in the same phagolysosomes. The study of the immunogenicity of various subcellular fractions revealed that the most active antibody formation is caused by the introduction of lysosomes into the body. The antigen is also found in cell membranes. Most of the processed antigen material secreted by macrophages has a stimulating effect on the proliferation and differentiation of T- and B-lymphocyte clones. A small amount of antigenic material can be stored in macrophages for a long time in the form chemical compounds, consisting of at least 5 peptides (possibly in connection with RNA).

In B-zones lymph nodes and spleen there are specialized macrophages (dendritic cells), on the surface of numerous processes of which many antigens are stored that enter the body and are transmitted to the corresponding clones of B-lymphocytes. In the T-zones of lymphatic follicles, interdigitating cells are located that affect the differentiation of T-lymphocyte clones.

Thus, macrophages are directly involved in the cooperative interaction of cells (T- and B-lymphocytes) in the body's immune responses.

The first study is always counting leukocyte formula(see the chapter "Hematological studies"). Both relative and absolute values ​​of the number of peripheral blood cells are evaluated.

Determination of the main populations (T-cells, B-cells, natural killers) and subpopulations of T-lymphocytes (T-helpers, T-CTLs). For the primary study of the immune status and detection of severe disorders of the immune system WHO recommended determination of CD3, CD4, CD8, CD19, CD16+56, CD4/CD8 ratio. The study allows to determine the relative and absolute number of the main populations of lymphocytes: T-cells - CD3, B-cells - CD19, natural killers (NK) - CD3-CD16++56+, subpopulations of T lymphocytes (T-helpers CD3+ CD4+, T-cytotoxic CD3+ CD8+ and their ratio).

Research method

Immunophenotyping of lymphocytes is carried out using monoclonal antibodies to superficial differential angina on the cells of the immune system, using laser flow cytofluorometry on flow cytometers.

The choice of the zone for analysis of lymphocytes is made according to the additional marker CD45, which is present on the surface of all leukocytes.

Conditions for taking and storing samples

Venous blood taken from the cubital vein in the morning, strictly on an empty stomach, into the vacuum system to the mark indicated on the test tube. K2EDTA is used as an anticoagulant. After sampling, the sample tube is slowly inverted 8-10 times to mix the blood with the anticoagulant. Storage and transportation strictly at 18–23°C in an upright position for no more than 24 hours.

Failure to comply with these conditions leads to incorrect results.

Interpretation of results

T-lymphocytes (CD3+ cells). An increased amount indicates hyperactivity of the immune system, observed in acute and chronic lymphocytic leukemia. An increase in the relative index occurs with some vyrus and bacterial infections at the onset of the disease, exacerbations of chronic diseases.

decline absolute quantity T-lymphocytes testifies to the insufficiency of cellular immunity, namely, the insufficiency of the cellular-effector link of immunity. It is detected in inflammations of various etiologies, malignant neoplasms, after trauma, operations, heart attack, smoking, taking cytostatics. An increase in their number in the dynamics of the disease is a clinically favorable sign.

B-lymphocytes (CD19+ cells) The decrease is observed with physiological and congenital hypogammaglobulinemia and agammaglobulinemia, with neoplasms of the immune system, treatment with immunosuppressants, acute viral and chronic bacterial infections, and the condition after removal of the spleen.

NK lymphocytes with CD3-CD16++56+ phenotype Natural killer cells (NK cells) are a population of large granular lymphocytes. They are able to lyse target cells infected with viruses and other intracellular antigens, tumor cells, and other cells of allogeneic and xenogeneic origin.

An increase in the number of NK cells is associated with the activation of anti-transplantation immunity, in some cases it is observed in bronchial asthma, occurs in viral diseases, increases with malignant neoplasms and leukemia, in the period of convalescence.

Helper T-lymphocytes with CD3+CD4+ phenotype An increase in absolute and relative amounts is observed in autoimmune diseases, may be with allergic reactions, some infectious diseases. This increase indicates the stimulation of the immune system to the antigen and serves as confirmation of hyperreactive syndromes.

A decrease in the absolute and relative number of T cells indicates a hyporeactive syndrome with a violation of the regulatory link of immunity, is a pathognomic sign for HIV infection; occurs in chronic diseases (bronchitis, pneumonia, etc.), solid tumors.

T-cytotoxic lymphocytes with CD3+ CD8+ phenotype An increase is detected in almost all chronic infections, viral, bacterial, protozoal infections. It is characteristic of HIV infection. The decrease is observed in viral hepatitis, herpes, autoimmune diseases.

CD4+/CD8+ ratio The study of the ratio of CD4+/CD8+ (CD3, CD4, CD8, CD4/CD8) is recommended only for monitoring HIV infection and controlling the effectiveness of ARV therapy. Allows you to determine the absolute and relative number of T-lymphocytes, T-helper subpopulations, CTL and their ratio.

The range of values ​​is 1.2–2.6. The decrease is observed in congenital immunodeficiencies (DiGeorge, Nezelof, Wiskott-Aldrich syndrome), viral and bacterial infections, chronic processes, exposure to radiation and toxic chemicals, multiple myeloma, stress, decreases with age, with endocrine diseases, solid tumors. It is a pathognomic sign for HIV infection (less than 0.7).

An increase in the value of more than 3 - in autoimmune diseases, acute T-lymphoblastic leukemia, thymoma, chronic T-leukemia.

The change in the ratio may be related to the number of helpers and CTLs in a given patient. For example, a decrease in the number of CD4+ T cells in acute pneumonia at the onset of the disease leads to a decrease in the index, while CTLs may not change.

For additional research and detection of changes in the immune system in pathologies requiring an assessment of the presence of an acute or chronic inflammatory process and the degree of its activity, it is recommended to include counting the number of activated T-lymphocytes with the CD3+HLA-DR+ phenotype and TNK cells with the CD3+CD16++56+ phenotype.

T-activated lymphocytes with CD3+HLA-DR+ phenotype A marker of late activation, an indicator of immune hyperreactivity. By the expression of this marker, one can judge the severity and strength of the immune response. Appears on T-lymphocytes after the 3rd day acute illness. With a favorable course of the disease, it decreases to normal. An increase in expression on T-lymphocytes can be associated with many diseases associated with chronic inflammation. Its increase was noted in patients with hepatitis C, pneumonia, HIV infection, solid tumors, autoimmune diseases.

ТNK lymphocytes with CD3+CD16++CD56+ phenotype T-lymphocytes bearing CD16++ CD 56+ markers on their surface. These cells have properties of both T and NK cells. The study is recommended as an additional marker for acute and chronic diseases.

Their decrease in peripheral blood can be observed in various organ-specific diseases and systemic autoimmune processes. An increase was noted in inflammatory diseases of various etiologies, tumor processes.

Study of early and late markers of T-lymphocyte activation (CD3+CD25+, CD3-CD56+, CD95, CD8+CD38+) additionally prescribed to assess changes in IS in acute and chronic diseases, for diagnosis, prognosis, monitoring of the course of the disease and ongoing therapy.

T-activated lymphocytes with CD3+CD25+ phenotype, IL2 receptor CD25+ is an early activation marker. The functional state of T-lymphocytes (CD3+) is evidenced by the number of expressing receptors for IL2 (CD25+). In hyperactive syndromes, the number of these cells increases (acute and chronic lymphocytic leukemia, thymoma, transplant rejection), in addition, their increase may indicate an early stage of the inflammatory process. In peripheral blood, they can be detected in the first three days of illness. A decrease in the number of these cells can be observed in congenital immunodeficiencies, autoimmune processes, HIV infection, fungal and bacterial infections, ionizing radiation, aging, heavy metal poisoning.

T-cytotoxic lymphocytes with CD8+CD38+ phenotype The presence of CD38+ on CTL lymphocytes was noted in patients with various diseases. Informative indicator for HIV infection, burn disease. An increase in the number of CTLs with the CD8+CD38+ phenotype is observed in chronic inflammatory processes, oncological and some endocrine diseases. During therapy, the rate decreases.

Subpopulation of natural killers with CD3-CD56+ phenotype The CD56 molecule is an adhesive molecule widely distributed in nervous tissue. In addition to natural killers, it is expressed on many types of cells, including T-lymphocytes.

An increase in this indicator indicates the expansion of the activity of a specific clone of killer cells, which have a lower cytolytic activity than NK cells with the CD3-CD16+ phenotype. The number of this population increases with hematological tumors (NK-cell or T-cell lymphoma, plasma cell myeloma, aplastic large cell lymphoma), chronic diseases, and some viral infections.

A decrease is noted in primary immunodeficiencies, viral infections, systemic chronic diseases, stress, treatment with cytostatics and corticosteroids.

CD95+ receptor is one of the receptors for apoptosis. Apoptosis is complex biological process necessary to remove damaged, old and infected cells from the body. The CD95 receptor is expressed on all cells of the immune system. It plays an important role in the control of the functioning of the immune system, as it is one of the receptors for apoptosis. Its expression on cells determines the readiness of cells for apoptosis.

A decrease in the proportion of CD95+ lymphocytes in the blood of patients indicates a violation of the effectiveness of the last stage of culling defective and infected own cells, which can lead to a relapse of the disease, chronicity of the pathological process, the development of autoimmune diseases and an increase in the likelihood of tumor transformation (for example, cervical cancer with papillomatous infection). Determination of CD95 expression has prognostic value in myelo- and lymphoproliferative diseases.

An increase in the intensity of apoptosis is observed in viral diseases, septic conditions, and the use of narcotic drugs.

Activated lymphocytes CD3+CDHLA-DR+, CD8+CD38+, CD3+CD25+, CD95. The test reflects functional state T-lymphocytes and is recommended for monitoring the course of the disease and monitoring immunotherapy for inflammatory diseases of various etiologies.

Activation markers - CD25, HLA-DR. appear on T-lymphocytes that are in the activation stage and can be used for diagnosis, assessment of the activity of immunopathological diseases, monitoring of treatment.

The level of CD3 + CD25 + , CD3 + HLA-DR + increased - activation of the immune response: acute or chronic inflammation, pneumonia, HIV infection, autoimmune and oncological diseases. Indicators decrease to normal with a favorable course of the disease.

The level of CD3 + CD25 + , CD3 + HLA-DR + is reduced - immunodeficiencies with impaired T-cell immunity.

T-helpers (CD3CD4)

CD3 + CD4 + elevated – acute infectious and inflammatory process, exacerbation of allergic and autoimmune diseases.

CD3 + CD4 + level reduced- congenital and acquired immunodeficiencies, HIV infection, Infectious mononucleosis, malignant neoplasms, long-term treatment with cytostatics, hormones, immunosuppressants.

T-cytotoxic (T-killers) (CD3CD8)

CD3 + CD8 + level reduced - primary and secondary immunodeficiencies, progressive malignant neoplasms, radiation therapy, long-term treatment with cytostatics, hormones, immunosuppressants.

Immunoregulatory index

In some diseases, the ratio is of diagnostic and prognostic value CD4/CD8 or immunological index (IRI). Norm: 1,2-2,5.

CD4/CD8 (IRI) elevated –autoimmune diseases, acute T-lymphoblastic leukemia, thymoma.

CD4/CD8 (IRI) decreased – viral infections (herpes, measles viral hepatitis, infectious mononucleosis, HIV/AIDS), chronic diseases tumors, multiple myeloma. The value of IRI less than 1 indirectly corresponds to immunodeficiency.

Natural killers (NK cells, CD16CD56)

NK cells are large granular lymphocytes that lyse target cells infected with viruses, intracellular pathogens, as well as mutant and tumor cells, without the specific recognition required for T-killers.

The level of NK cells (CD3 + - CD16 + CD56 +) is increased - acute inflammatory process bronchial asthma, malignant neoplasms, leukemia.

NKT cells (CD3CD16CD56)

HKT cells have receptors for both NK cells and T lymphocytes. Carry out a regulatory function by synthesizing cytokines.

Level (CD3 + CD16 + CD56 +) increased - in acute severe inflammatory processes, oncological diseases.

The level (CD3 + CD16 + CD56 +) is reduced - with autoimmune diseases.

B-lymphocytes (CD19,CD20)

Level CD19 + ,CD20 + With low - hypo- and agammaglobulinemia (congenital and acquired); lymphocytic leukemia, non-B-cell type lymphoma; splenectomy; taking immunosuppressants.

Immunodiagnostics of violations of the synthesis of immunoglobulins

Determination of the content of immunoglobulins is important in the diagnosis and clinical monitoring of congenital immunodeficiencies, monoclonal gammopathy, infections and autoimmune diseases. Methods for the determination of immunoglobulins: radial immunodiffusion according to Mancini, nephelometry. The level of IgE is determined by ELISA.

Immunoglobulin M (IgM)- globulin protein molecular weight 900 kDA, synthesized by B-lymphocytes mainly during the initial contact with the antigen.

Immunoglobulin M is formed 3-6 days after interaction with the antigen and provides the main protection of the body during bacteremia. Does not penetrate the vascular wall, the placental barrier.

The level of IgM is reduced - primary immunodeficiencies with impaired humoral link, protein-losing gastroenteropathy, burn disease, IgG or IgA-type multiple myeloma, splenectomy, immunosuppressive therapy.

Immunoglobulin G (IgG) - a protein of globulin nature with a molecular weight of 150 kDA, synthesized by B-lymphocytes in response to an antigenic stimulus. In case of primary infection, it appears after 8-14 days and protects the body from pathogens and their toxins. IgG penetrates through the vascular wall and the placenta.

The level of IgG is increased - protracted, chronic and recurrent infections, autoimmune diseases ( rheumatoid arthritis, SLE, Sjögren's disease), liver diseases (hepatitis, cirrhosis), malignant neoplasms, IgG-type multiple myeloma, benign paraproteinemia.

The level of IgG is reduced - primary immunodeficiencies with impaired humoral link, protein-losing gastroenteropathy, burn disease, nephrotic syndrome, IgA or IgM-type multiple myeloma, splenectomy, immunosuppressive therapy.

Immunoglobulin A (IgA)- a protein of blood serum and secretions of mucous membranes with a molecular weight of 160 kDA, synthesized by B-lymphocytes in response to antigenic exposure. Appears 15-21 days after contact with the antigen, prevails in mucosal secretions (saliva, lacrimal fluid, nasal secretions, sweat, bronchial secretions), where it provides protection from pathogens, pollen and food allergens.

The level of IgA is increased - chronic infections of the digestive system and respiratory tract, tumors of the lower gastrointestinal tract, autoimmune diseases (at an early stage), IgA-type multiple myeloma, liver diseases (hepatitis, cirrhosis), osteosarcoma, myeloma, cystic fibrosis.

The level of IgA is reduced - congenital deficiencies with a violation of the humoral link, chronic and recurrent diseases of the respiratory tract, protein-losing gastroenteropathy, burn disease, nephrotic syndrome, multiple myeloma of IgG and IgM-type, splenectomy, immunosuppressive therapy.

Immunoglobulin E (IgE) - protein of blood serum and secretions of mucous membranes with a molecular weight of 200 kDa. Synthesized by B-lymphocytes in response to environmental antigens (house dust, plant pollen, infectious agents, foodstuffs, etc.) and cause allergic reactions immediate type.