Immunological memory Inhabitants: 46 000 Area: 1400 km2 1781: Measles epidemic in the Faroe Islands No measles for 65 years 1846: Measles epidemic (Peter Panum, Danish physiologist) Those individuals, who were older than 65 years and were infected in 1781 did not become sick, but some elderly people got the infection – resistance to virus Life-long protection can be induced against some viruses Presence of the virus is not needed for the maintenance of immunological memory Immunological memory is harnessed by vaccination

DEVELOPMENT OF PRIMARY – EFFECTOR – MEMORY T AND B LYMPHOCYTES IN THE COURSE OF ANTIGEN – SPECIFIC IMMUNE RESPONSES current infection prospective infection

Retention of vaccina-specific antibodies and T cells after vaccination against smallpox virus. Specific anti-vaccinia antibodies continue to be made for as long as 75 years after the last exposure to vaccinia virus, the smallpox surrogate that is used for vaccination (top panel). The numbers represent international units (IU) of antibody, a standardized way of measuring an antibody response. Many vaccinated individuals retain populations of vaccinia-specific CD4 T cells and CD8 T cells (bottom panel). Only small differences are observed for individuals who received one (blue bars) or two (pink bars) vaccinations.

Specificity is a prerequisit of memory THE TWO ARMS OF THE IMMUNE SYSTEM Monocytes, Macrophages, Dendritic cells, Granulocytes, NK cells and Complement components Monocytes, Macrophages, Dendritic cells, Granulocytes, NK cells and Complement components B and T cells Defense against microbes is mediated by the early reactions of innate immunity and the later responses of adaptive immunity. Innate immunity (also called natural or native immunity) provides the early line of defense against microbes. It consists of cellular and biochemical defense mechanisms that are in place even before infection and are poised to respond rapidly to infections. These mechanisms react to microbes and to the products of injured cells, and they respond in essentially the same way to repeated infections. The principal components of innate immunity are (1) physical and chemical barriers, such as epithelia and antimicrobial chemicals produced at epithelial surfaces; (2) phagocytic cells (neutrophils, macrophages), dendritic cells, and natural killer (NK) cells; (3) blood proteins, including members of the complement system and other mediators of inflammation; and (4) proteins called cytokines that regulate and coordinate many of the activities of the cells of innate immunity. The mechanisms of innate immunity are specific for structures that are common to groups of related microbes and may not distinguish fine differences between microbes.   In contrast to innate immunity, there are other immune responses that are stimulated by exposure to infectious agents and increase in magnitude and defensive capabilities with each successive exposure to a particular microbe. Because this form of immunity develops as a response to infection and adapts to the infection, it is called adaptive immunity. The defining characteristics of adaptive immunity are exquisite specificity for distinct molecules and an ability to "remember" and respond more vigorously to repeated exposures to the same microbe. The adaptive immune system is able to recognize and react to a large number of microbial and nonmicrobial substances. In addition, it has an extraordinary capacity to distinguish between different, even closely related, microbes and molecules, and for this reason it is also called specific immunity. It is also sometimes called acquired immunity, to emphasize that potent protective responses are "acquired" by experience. The main components of adaptive immunity are cells called lymphocytes and their secreted products, such as antibodies. Foreign substances that induce specific immune responses or are recognized by lymphocytes or antibodies are called antigens. Innate and adaptive immune responses are components of an integrated system of host defense in which numerous cells and molecules function cooperatively. The mechanisms of innate immunity provide effective initial defense against infections. However, many pathogenic microbes have evolved to resist innate immunity, and their elimination requires the more powerful mechanisms of adaptive immunity. There are many connections between the innate and adaptive immune systems. The innate immune response to microbes stimulates adaptive immune responses and influences the nature of the adaptive responses. Conversely, adaptive immune responses often work by enhancing the protective mechanisms of innate immunity, making them capable of effectively combating pathogenic microbes. Specificity is a prerequisit of memory

The role of adaptive immune system : Primary immune response? Slow, effectitity? 2. During reinfection! Immediate/quick, effective

B cell memory

B cell memory: Quicker response Increase in the number of specific B cells The amount of antibody are higher Higher affinity antibodies (‘more specific’) Isotype switch In case of T dependent B cell activation

is provided mainly by T dependent response via GC B cells B cell memory is provided mainly by T dependent response via GC B cells Memory B cells proliferation and differentiation to plasma cell, protection in 3-5 days and Long-lived plasma cells Constitutive antibody production, immediate protection

B cell memory is provided by: Memory B cells Express cell surface antobody (BCR), went through somatic mutation, affinity maturation, izotype switch. Localized mainly in the circulation upon re-activation proliferate and differentiate to plasma cell Long-lived plasma cells produce antibody Localized mainly in the bone marrow Immediate neutralization, initiation of effector functions

Comparision of memory B cells and long-lived plasma cells Memória B cells Long-lived plasma cells lifespan years Reaction time days immediate antibody Cell surface (BCR) soluble Antigen dependence Activation only in the presence of antigen Non-sensitive, constitutive antibody production proliferation Upon recognition of antigen No proliferation

T cell dependent B cell activation may results in: Short lived plasma cells (life span 10-20 days), localised mainly in the secondary lymphoid organs, it has role in the current immune response. (and/or block the short term reinfection.) Long-lived palma cell c.Memory B cell.

during the primary immunization ”Short term memory” during the primary immunization

Extrafollicular plasma cell Initiation of GC reaction Extrafollicular B cell activation generates quick, but less efficient response The initial interaction of activated helper T cells with antigen-specific B cells at the edge of the follicle induces some proliferation and differentiation of B cells. Extrafollicular foci of T-dependent B cell activation are generated relatively early in an immune response. Each such focus may produce 100 to 200 antibody-secreting plasma cells. Plasma cells that are generated in extrafollicular foci are mostly short-lived, and these cells do not acquire the ability to migrate to distant sites such as the bone marrow. The small amount of antibody produced in these foci may contribute to the formation of immune complexes (containing antigen, antibody, and perhaps complement) that are trapped by follicular dendritic cells in lymphoid follicles. High affinity B cells differentiate to plasma cells at the beginning of the response. Low affinity entering the GC go trough affinity maturation, increase the affiinity finaly exceed the original ‘high ‘ affinity ones. Extrafollicular plasma cell or Initiation of GC reaction 13

Short lived plasma cells are created mainly in the extrafolliculare B cell activation in a T-dependent way.

Presence of specific antibodies during primary and secondary immune responses protects against repeated infections Antibodies produced during the primary immune response protects against short term re-infection by neutralization and opsonization. (a few days- a few weeks)

Comparison of primary and memory B cell response

B cell memory: Quicker response Higher affinity antibodies (‘more specific’) Increase in the number of specific B cells, the amount of antibody are higher Isotype switch In case of T dependent B cell activation

Time requirements of primary and memory response Time consuming steps of primary response: 1. Formation of immune complexes 2. Proliferation and selection of naiv B cells 3. Antigen presentation of B cells 4. Differentiaition of B cells to plasma cells.

Time consuming steps of primary response 1: I. Formation of immune complexes Antigen entry to the GC: Soluble antigens, generally smaller than 70 kD, Antigen > 70 kDa antigen, mainly immunkomplexes can be transproted. Conduits see the previous slide Capture of immunkomplexes is mediated by complement (and Fc) receptors

Time consuming steps of primary response 1: II. Formation of immune complexes Immune complexes are trapped on the surface of FDCs B Memory B cell apoptosis T CD40 CD40L Follicular dendritic cell (FDC) FcR CD21 Ag No Ag CD21--- CR2

immuncomplex---which cell produced the antibody? Primary response immuncomplex---which cell produced the antibody? (natural antibodies, extrafollicular B cells?? Complement components alone?) During Memory response: Antibodies produced by long-lived plasma cells Immediate protection (neutralization) Accelerated immunocomplex formation

Time consuming steps of primary response 2 : Proliferation and selection of naiv B cells Proiferation in the germinal center. During primary response multiplication of a few clones. Germinal centers, in which specialized follicular helper T (TFH) cells trigger B cells to undergo numerous changes, appear a few days later. B cells that have been activated by T helper cells at the edge of a primary follicle migrate into the follicle and proliferate, forming the dark zone of the germinal center. Germinal center B cells undergo extensive isotype switching. Somatic hypermutation of Ig V genes occur in these B cells, and they migrate into the light zone, where they encounter follicular dendritic cells displaying antigen and TFH cells. B cells with the highest affinity Ig receptors are selected to survive, and they differentiate into antibody-secreting or memory B cells. The antibody-secreting cells leave and reside in the bone marrow as long-lived plasma cells, and the memory B cells enter the recirculating pool. 22

During Memory response: Earlier engagement of antigen (immune complexes) leads to earlier initiation of the proliferation Higher number of clones initiate proliferation (128 in stead of 4 clones at the begining of response– 5 cell division –2-3 days benefit)

Time consuming steps of primary response 3: Antigen presentation of B cells High affinity B cells can present the antigens for T cells Germinal centers, in which specialized follicular helper T (TFH) cells trigger B cells to undergo numerous changes, appear a few days later. B cells that have been activated by T helper cells at the edge of a primary follicle migrate into the follicle and proliferate, forming the dark zone of the germinal center. Germinal center B cells undergo extensive isotype switching. Somatic hypermutation of Ig V genes occur in these B cells, and they migrate into the light zone, where they encounter follicular dendritic cells displaying antigen and TFH cells. B cells with the highest affinity Ig receptors are selected to survive, and they differentiate into antibody-secreting or memory B cells. The antibody-secreting cells leave and reside in the bone marrow as long-lived plasma cells, and the memory B cells enter the recirculating pool. 24

During Memory response: memory B cells –already selected, high affinity cells- are more effective antigen presenting cells

Time consuming steps of primary response 4: Differentiaition of B cells to plasma cells. High affinityB cells differentiate to plasma cells. (or to memory cells) Germinal centers, in which specialized follicular helper T (TFH) cells trigger B cells to undergo numerous changes, appear a few days later. B cells that have been activated by T helper cells at the edge of a primary follicle migrate into the follicle and proliferate, forming the dark zone of the germinal center. Germinal center B cells undergo extensive isotype switching. Somatic hypermutation of Ig V genes occur in these B cells, and they migrate into the light zone, where they encounter follicular dendritic cells displaying antigen and TFH cells. B cells with the highest affinity Ig receptors are selected to survive, and they differentiate into antibody-secreting or memory B cells. The antibody-secreting cells leave and reside in the bone marrow as long-lived plasma cells, and the memory B cells enter the recirculating pool. 26

During Memory response: High affinity B cells - practically memory B cells - differentiate to plasma cells. (Correlation between affinity and time.)

B cell memory: Quicker response Higher affinity antibodies (‘more specific’) Increase in the number of specific B cells, the amount of antibody are higher Isotype switch In case of T dependent B cell activation

ANTIGEN SPECIFIC B CELLS ACQUIRE ANTIGEN FROM THE SURFACE OF FDCs Complement fragments High-affinity B cell Fc receptors FDC Complement receptors TÁMOP-4.1.1.C-13/1/KONV-2014-0001

During memory response High affinty antibodies, produced by long lived plasma cells High-affinity B cell Fc receptors FDC Complement receptors TÁMOP-4.1.1.C-13/1/KONV-2014-0001

During Memory response Memory B cells (selected, high affinity cells) compete with naiv B cells and memory cells in GC cycles In consecutive responses the new clones must outcompete the existing ones, due to it the affintiy increses in all the response

B cell memory: Quicker response Higher affinity antibodies (‘more specific’) Increase in the number of specific B cells, the amount of antibody are higher Isotype switch In case of T dependent B cell activation

During Memory response: Higher number of initiating clones Higher affinity clones are more effective in antigen presentation resulting in more intense T cell help leading to survival and plasma cell differentiation

B cell memory: Quicker response Higher affinity antibodies (‘more specific’) Increase in the number of specific B cells, the amount of antibody are higher Isotype switch In case of T dependent B cell activation

During Memory response During GC reactions the optimal „Fc region” effector functions are created. (driven by the cytokine enviroment)

Effect of age on the expressed B cell repertoire

PRODUCTION OF IMMUNOGLOBULINS Fetus/newborn are not protected against new pathogens by B cells!

a a a DEVELOPMENT OF THE ADAPTIVE IMMUNE SYSTEM B-CELLS The adult immune system relies more and more on memory B cells Transient B cells (T1/T2) mature B cell  memory B cell CD24 New born 1 year 5 year memory memory memory CD38

DEVELOPMENT OF THE ADAPTIVE IMMUNE SYSTEM B-CELLS 17 year 28 year 59 year CD24 memory memory memory CD38

Consequenses: Less effecitve adaptive immunity in newborn Reactivity to new infections may decline in ages Fetus is protected only against pathogens, which generated memory response in the mother. (innate immunity is active in all kind of infection)

IMMUNOLOGICAL MEMORY – B CELLS SUMMARY I Germinal Center reaction B cell proliferation Somatic hypermutation Affinity maturation Memory B cells Perviously activated Passed through affinity maturation Present in the circulation Rapid proliferation and differentiation to plasma cell upon re-activation or entry to the GC reaction again Plasma cells Provides serological memory by pre-existing neutralizing Abs to pathogens and/or toxins B B FDC B FDC B T B B B B B B plasma cell B B B B B B – T cell collaboration T B

IMMUNOLOGICAL MEMORY – B CELLS SUMMARY I 42

Goal(s) of memory response: To recognize the reappearance of pathogen Flexibility, to recognize the new variants of the pathogens 1. Long lived plasma cells produce high affinity antibodies (non flexible, but effective) 2. Memory B cells are relativelly low affinity, flexible

Extrafollicular B cell activation Handicap in competition Extrafollicular B cells can produce memory cells, no somatic mutation --- low affinity Handicap in competition The initial interaction of activated helper T cells with antigen-specific B cells at the edge of the follicle induces some proliferation and differentiation of B cells. Extrafollicular foci of T-dependent B cell activation are generated relatively early in an immune response. Each such focus may produce 100 to 200 antibody-secreting plasma cells. Plasma cells that are generated in extrafollicular foci are mostly short-lived, and these cells do not acquire the ability to migrate to distant sites such as the bone marrow. The small amount of antibody produced in these foci may contribute to the formation of immune complexes (containing antigen, antibody, and perhaps complement) that are trapped by follicular dendritic cells in lymphoid follicles. High affinity B cells differentiate to plasma cells at the beginning of the response. Low affinity entering the GC go trough affinity maturation, increase the affiinity finaly exceed the original ‘high ‘ affinity ones. 44

Memory B cells during memory response can be Extrafollicular B cells Memory B cells Long-lived plasma cells The initial interaction of activated helper T cells with antigen-specific B cells at the edge of the follicle induces some proliferation and differentiation of B cells. Extrafollicular foci of T-dependent B cell activation are generated relatively early in an immune response. Each such focus may produce 100 to 200 antibody-secreting plasma cells. Plasma cells that are generated in extrafollicular foci are mostly short-lived, and these cells do not acquire the ability to migrate to distant sites such as the bone marrow. The small amount of antibody produced in these foci may contribute to the formation of immune complexes (containing antigen, antibody, and perhaps complement) that are trapped by follicular dendritic cells in lymphoid follicles. High affinity B cells differentiate to plasma cells at the beginning of the response. Low affinity entering the GC go trough affinity maturation, increase the affiinity finaly exceed the original ‘high ‘ affinity ones. 45

T cell memory: 47

Activation of helper and cytotoxic T cell equally result in memory cell formation

T cell memory: Quicker response Increase in the number of responding cells

T cell memory response is provided by : Long-lived effektor T cells –quick reaction and Central memory T cells –proliferation, differentiation

Naive T cells (either CD4+ or CD8+) can differentiate to Effector T cells (role in the primary response) Effector memory cells Central memory cells

II. Long-lived plasma cells Analogy with B cells: I. Memory B cells proliferation and differentiation to plasma cell upon re-activation or entry to the GC reaction again and II. Long-lived plasma cells Plasma cells generated during GC reaction migrate to bone marrow and survive for years, producing antibody. Immediate protection. Much of circulating IgG,IgA is produced by long-lived plasma cells, provides initial protection I. Central memory T cells: In the lymph node (DC-mediated presentation), requires proliferation and differentiation to effector cells II. Effector memory T cells: Periferial activation, limited proliferation Quick response (not immediate requires antigen presentation)

Citokines/Cytotoxicity Naive T Effector T Citokines/Cytotoxicity AICD Naive T Effector memory T Effector T Cytokines/cytotoxicity ANTIGEN/SITE OF INFECTION Central memory T CCR7 L selectin Effector T Citokines/cytotoxicity PERIPHERAL LYMPHOID ORGANS PERIPHERAL TISSUES Skin dermis, gut lamina propria, alveolar space Tissue-specific migration

Functional differences between lymphoid tcm cells and tissue-resident TEM cells The localization of effector memory T (TEM) cells in peripheral tissues before infection is clearly an important factor in their ability to provide protection from a secondary infection. However, several studies have also shown that TEM cells and central memory T (TCM) cells have different effector functions in response to antigen stimulation51,52 (see the figure). Although both subsets of memory T cells produce large amounts of effector cytokines such as interferon‑γ (IFNγ) and tumour‑necrosis factor (TNF) after antigen stimulation, a greater frequency of TCM cells also produce interleukin‑2 (IL‑2), which could increase their ability to proliferate in response to antigen94. By contrast, TEM cells have more potent lytic activity ex vivo compared with TCM cells. This is probably due to the increased expression of perforin by both resting and activated TEM cells95, and because resting tissue‑resident memory T cells (that is, TEM cells) can maintain the expression of mRNA transcripts that encode cytolytic proteins96. The presence of this pre‑formed mRNA enables TEM cells to express cytolytic proteins, such as granzyme B, more rapidly, thereby increasing their ability to rapidly kill infected cells. In addition, a small fraction of TEM cells can express the low‑affinity Fc receptor for IgG IIIa (FcγRIIIa) (not shown), which allows them to directly mediate antibody‑dependent cell‑mediated cytotoxicity97. Proliferation Cytotoxicity cytotoxicity Woodland DL & Kohlmeier JR 2009 Nat Rev 9:153

Longevity of memory cells are provided by: 1. increased levels of anti-apoptotic proteins, 2. memory cells undergo slow proliferation, and thus able to self-renew most important cytokine for the maintenance of memory CD4+ and CD8+ T cells is IL-7 (and IL-15) BUT do not require the presence of the antigen!

CD4+ Memory T cells are heterogeneous some CD4+ memory T cells may be derived from precursors before commitment to the TH1, TH2, or TH17 phenotype thus can differentiate into any of these subsets. (flexibility?) Other memory T cells may be derived from fully differentiated TH1, TH2, or TH17 effectors and retain their respective cytokine profiles on reactivation.

Comparison of primary and memory T cell response

T cell memory: Quicker response Increase in the number of responding cells

Time consuming steps of naive T cell activation: 1 .Cross-presentation 2. Antigen presenting cells migrate to the secondary lymphoid organs 3 .Specific naiv T cells proliferate and differentiate 4. Effector T cells migrate to the infected tissue

Primary T cell activation I It takes ??? days Crosspresentation ---antigen must localize into DCs

respond to antigens presented by a wide range of APCs During memory response effector memory cells do ot require cross-presentation, any somatic cell is able to activate them (T cell reaction (may) accelerate the process of cross presentation ) memory T cells are less dependent on costimulation than are naive cells respond to antigens presented by a wide range of APCs

Primary T cell activation II It takes 1day Activation and migration of DCs

During memory response effector memory cells are located in the periphery, these cells can recognize the antigen without transport. (but requires antigen presentation) differences in the expression of adhesion molecules and chemokine receptors.

Primary T cell activation III It takes 5-7day Clonal proliferation of naiv T cells

During memory response effector memory cells merely proliferate Higher number of central memory T cell clones initiate proliferation Do not, or merely require costimulation

Primary T cell activation IV Migration of effector T lymphocytes

Effector memory T cells are localized in various compartments During memory response Effector memory T cells are localized in various compartments Central memory T cells migrates to the infected tissues following proliferation and differentiation GENERAL ENTRY SITES LIMITED ENTRY SITES Brain Alveoli Peritoneum Lamina propria Skin Lung parenchyma Lymph node Spleen Liver Bone marrow General : CD43 LFA-1 P-selectin VLA-4 Tissue Specific: αVβ7integrin----Gut αVβ7integrin---Lung CD62L -Lymph node

T cell memory: Quicker response Increase in the number of responding cells

During memory response Pre-existing effector memory cells Higher number of central memory T cell clones initiates the proliferation

Maintained by cytokines: Summary: IMMUNOLOGICAL MEMORY MEDIATED BY T LYMPHOCYTES Naive T cell Effector T cell 2X107 cytokine production cytotoxicity Central Memory T cell Effector T cell Maintained by cytokines: IL-7, IL-15 Previously activated, partially differentiated cell type Circulating CCR7+ cells in blood, lymphoid tissues High proliferation rate induced by activation signals Rapid differentiation to effector cells Half of the 1012 peripherial T cells are naiv, half of it are memory with: naiv cells have 2*107 specificities Memory T cells have only 105 antigen specificities 2X105 Effector Memory T cell Effector T cell Previously activated, partially differentiated cell type Closest to the effector state Circulating CCR7- cells in blood and tissues Slow proliferation, rapid effector functions

IMMUNOLOGICAL EXPERIENCE AGE THYMUS PERIPHERY M E O R Y N A I V E IMMUNOLOGICAL EXPERIENCE In individuals older than 50 years of age or so, half or more of circulating T cells may be memory cells.

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