III Week 6

How does activation and differentiation of naive T cells occur?

- APCs deliver 3 kinds of signals for clonal expansion and differentiation - Antigen:MHC complex binding to the T cell receptor (TCR) on naïve T cells transmits a signal to T cell that the antigen has been recognised (Signal 1) - Signal 1 alone is not adequate for T cell activation - T cell activation require a 2nd signal (Signal 2) which is known as a co-stimulatory signal delivered by the same APC - CD28 dependent co-stimulation triggers T cells to enter cell cycle and induces synthesis of IL-2 and its receptor IL-2R - IL-2 drives T cell proliferation - clonal expansion • These mechanisms form part of the normal and abnormal functions of the immune system • May be able to modify these signals - important in the tolerance mechanisms

Hyperacute Graft Rejection - Cause - Process

- Caused by antibodies that react with endothelium - Recipient already has Abs against Ags in the graft - Recipients already have Ab to donor antigens, which are often blood group antigens or HLA Ag - When the donor organ is grafted into such recipients, these Ab bind to vascular endothelium in the graft, initiating the complement and clotting cascades - Can lead to destruction of the endothelial cells - Complement can cause recruitment, MAC formation, destroying the endothelial cells - Platelets and acute phase proteins can be recruited - Blood vessels become blocked - less oxygenation - tissue dies - platelets can come along and start to create clots - No specific role of T cells, but antibodies play an important role - Blood vessels in the graft become obstructed by clots and leak, causing hemorrhage of blood into the graft.

How can graft versus host disease be beneficial?

- GVHD can have effects that are crucial to the success of the transplant (Sometimes beneficial): o In leukaemia the therapeutic effect of HSC transplant is mostly due to GVHD or more accurately Graft versus-leukaemia effect → Donor T cells in the allogeneic preparations of HSCs recognize minor histocompatibility antigens expressed by the leukemic cells and kill the leukemic cells

The Major Histocompatibility Complex - other name? genes involved in encoding of chains? where are these genes located?

- HLA (human leucocyte antigens) - Structure: α chain of the MHC class I and both the α and β chains of the MHC class II cell surface receptors are encoded by the MHC. The MHC genes are located on the short arm of chromosome 6

Common organs transplanted and reason for transplantation

- Kidney in end stage renal failure - Heart in terminal cardiac failure - Heart & Lung for pulmonary hypertension and cystic fibrosis - Liver in cirrhosis, cancer, biliary atresia - Cornea in dystrophy, keratitis - Pancreas or islets cells in diabetes - Bone marrow in leukaemia, congenital abnormalities - Small bowel in cancer (e.g. if they have large amounts of their bowel resected) - Skin for burns

What role to T cells play in the development of self tolerance?

- T cells are considered to be mainly responsible for self tolerance - T cells that encounter self antigens during thymic development are clonally deleted - Without T cell help B cells are unable to produce autoantibodies - T cells escaping this process may be regulated by other cells in the periphery e.g. regulatory T cells - T cells cannot respond to self antigens in association with class I MHC molecules on non-haematogeneous cells

Antigens looked at when doing MHC matching between recipient and donor and the impact of these

- The results reported for compatibility will generally include six numbers: Two HLA- A antigens, two HLA-B antigens and two HLA-DR antigens, as a minimum e.g. ϖ Patient A3,32 B7,37 DRB1*01:01, 15:01 ¬ B1 = beta 1 chain ϖ Donor A3,32 B7,37 DRB1*01:01, 15:01 ϖ HLA-DP, HLA-DQ may also be matched - Should try for 6 HLA molecules matched between donor and recipient - can match other HLA molecules as well (e.g. HLA-DP and -DQ) - example above is a 6/6 match - If you have 5 of 6 matches, it doesn't mean you won't get the organ, but the chance of rejection is greater • the major impact in graft loss comes from the effects of HLA-B and -DR - effects of HLA-DR mismatches are the most important in first 6 months post Tx, HLA-B effect emerges in first 2 years, HLA-A mismatches have a deleterious effect on long-term graft survival ϖ Mismatched -DR - most likely to see rejection in the first 6 months • often use T cell immunosuppression to suppress the unwanted T cell responses - match MHC

BMT is used for...?

- Therapy in some tumour derived from haematopoietic cells (e.g. certain leukaemias and lymphomas) - Cure for some primary immunodeficiencies (replacing genetically defective HSCs with normal ones) - Cure for some inherited blood cell disorders (e.g. severe forms of thalassemia)

Acute reaction - time, effector mechanisms?

- Time: Days-Weeks (7-21dd) - Effector Mechanisms: Activation of naive T cells = 1st set skin graft rejection • Acute - T cells being primed against the antigens - first set skin graft rejection

Hyperacute reaction - time, effector mechanisms?

- Time: Min-Hrs - Effector Mechanisms: Preformed anti-donor Ab (ABO & MHC), Ab binding to endothelial cells, Activation of complement cascade • Hyperacute - happens very quickly after a graft is planted in the recipient - only possible if there are mediators there ready to attack the graft straight away (Antibodies) - Abs will bind to graft antigens and will cause the graft to be rejected - Graft is often destroyed because of the occlusion of arteries - Pre-exisitng alloantibodies against blood group antigens and polymorphic MHC antigens can cause rapid rejection of transplanted organs - Complement-dependent reaction - Can occur within minutes of transplantation - Antibodies of the ABO type can bind to all tissues, not just red blood - Antibodies against other antigens can be produced in response to a previous transplant or a blood transfusion. - All such preexisting antibodies can cause rapid rejection of vascularized grafts because they react with antigens on the vascular endothelial cells of the graft and initiate the complement and blood clotting cascades. - The vessels of the graft become blocked, or thrombosed, causing its rapid destruction. Such grafts become engorged and purple-colored from hemorrhaged blood, which becomes deoxygenated - This problem can be avoided by ABO-matching as well as cross-matching donor and recipient. - Cross-matching involves determining whether the recipient has antibodies that react with the white blood cells of the donor. Antibodies of this type, when found, have hitherto been considered a serious contraindication to transplantation of most solid organs, because in the absence of any treatment they lead to near-certain hyperacute rejection. - Get inflammatory responses due to complement binding

Chronic reaction - time, effector mechanisms?

- Time: Months-Years - Effector Mechanisms: Multifactoral: Ab, immune complexes T cell activation, immunosuppressants, original disease process Chronic - reject the graft over a long period of time - more complex process - Also caused by macrophages being recruited as chronic inflammatory cells, releasing cytokines which damage the graft

Accelerated reaction - time, effector mechanisms?

- Time:Days (2-5dd) - Effector Mechanisms: Reactivation of sensitised T cells Cell & humoral immune responses = 2nd set skin graft rejection • Accelerated - already have memory cells against the antigens - have sensitised T cells that react to a donor molecule - Skin that has previously rejected a graft ir regrafted with skin from the same donor - Skin from a 3rd party donor grafted onto the same recipient at the same time doesn't show this faster response, but follows a first-set rejection course

Acquired immunological tolerance

- Tolerance arising as a result of administration of exogenous Ag

In the Sensitisation Stage there are 2 ways alloantigens are presented on the transplanted donor organ to the recipient's T lymphocytes:

1) Direct recognition of a grafted organ: - Mediated by T cells whose TCR have specificity for the allogeneic MHC class I or class II molecule in combination with peptide - Donor graft APCs have a high density of allo- MHC molecules and are capable of directly stimulating the recipient's T cells > proliferate - Before naive alloreactive T cells can develop into effector T cells that cause rejection, they must be activated by antigen-presenting cells that express both the allogeneic MHC and co-stimulatory molecules. - Organ grafts carry with them antigen-presenting cells of donor origin, sometimes called passenger leukocytes, and these are an important stimulus to alloreactivity. - This route for sensitization of the recipient to a graft seems to involve donor antigen- presenting cells leaving the graft and migrating to secondary lymphoid tissues of the recipient, including the spleen and lymph nodes, where they can activate those host T cells that bear the corresponding T-cell receptors. - Because the lymphatic drainage of solid organ allografts is interrupted by transplantation, migration of donor antigen-presenting cells occurs via the blood, not lymphatics. The activated alloreactive effector T cells can then circulate to the graft, which they attack directly - Largely responsible for acute rejection ϖ Especially when MHC mismatches mean that the frequency of directly allo- reactive recipient T cells is high. ϖ A direct cytotoxic T-cell attack on graft cells can be made only by T cells that recognize the graft MHC molecules directly. Process: - Host T cells (Both CD4 and CD8) directly recognise the foreign MHC molecules that are expressed on the graft DC. - CD4 T cells then proliferate and produce cytokines that induce tissue damage as well as assisting in B cells production of Abs. - CD8 T cells differentiate into cytotoxic T cells that attack the graft antigens directly 2) Indirect recognition of graft - Mediated by T cells whose TCR specific for allogeneic peptides that are derived from the grafted organ. - Proteins from the graft (including MHC molecules) are processed by the recipient's APC and are therefore presented by self (recipient) MHC class I or class II molecule o Recipient's DC is taking up the foreign Ag from the graft and MHC becomes the foreign peptide - can activate recipient T cell o Uptake of allogenic proteins by the recipient's own APCs and their presentation to T cells by self-MHC molecules o Can present peptides derived from both the foreign MHC molecules and minor histocompatibility antigens - NOTE: allogeneic MHC present on any cell in the graft (not just the DCs) Process: --> Host DCs recognises, takes up and processes the MHC complex into peptides and then host CD4 T cells recognise donor MHC peptides. --> CD4 T cells then proliferate and produce cytokines that induce tissue damage as well as assisting in B cells production of Abs.

Requirements for bone marrow transplant (BMT)

1) HLA Matching a. Matching of MHC class I & II genes between donor and recipient essential to prevent graft rejection and graft versus host disease (GVHD) b. Serological techniques: i. Microcytotoxicity assay - using standard typing sera c. Molecular techniques: RFLP, PCR-sequence specific oligonucleotide (PCR-SSO), PCR-sequence specific primers (PCR-SSP), Sequence based typing (SBT) - can identify HLA type based on the DNA sequence and match these 2) Cross Matching a. To determine whether Ab are present in the recipient's serum that may cross-react with the donor cell surface Ag (to prevent hyperacute rejection) 3) Mixed Lymphocyte Culture (MLC) a. Mix lymphocytes from two individuals, different histocompatibility antigens on the surface of donor lymphocytes will activate the recipient lymphocytes and vice versa. DNA synthesis> proliferation of lymphocytes measure incorporation of radioactively labelled thymidine - where a lymphocyte proliferation assay is used

The principles of transplantation

1. Graft between genetically identical individuals - accepted 2. Graft between non-identical individuals - rejected 3. 1st & 2nd set skin graft rejection - 1st set rejection - rejection of first graft - 2nd set rejection faster because there are memory cells (especially memory T cells) 4. Enlargement of lymph nodes at the time of rejection 5. Transfer of 2nd set skin graft rejection by lymphocytes 6. Specificity of 2nd set skin graft rejection 7. The central role of T cells in rejection

3 ways of immune intervention in transplantation and ultimate goal

1. Immunosuppression of recipient & histocompatibility matching 2. Induction of specific tolerance (to transplant Ags) 3. Pretreatment of allograft prior to transplant (e.g. removing certain types of cells or blocking antigen presentation) The ultimate goal: Development of a specific immunosuppressive protocol directed towards the immune response to the allograft • Consequent of this is that the patient may be more susceptible to infectious diseases or more likely to get cancers (as T cells help fight against malignancies)

Polymorphism:

>200 alleles of some class I and class II genes They are some of the most polymorphic molecules between different individuals • Foreignness of the MHC is a limiting factor in finding transplants that are compatible • MHC II mainly expressed on APCs

Autoantigen

A self antigen to which the immune system makes a response. Autologous

Acute and severe rejection symptoms of kidney graft rejection

Acute symptoms - • Organ function decreases • Decreased urine production • Swelling of hands and ankles • Main clinical indicator is increasing serum creatinine level above baseline value Severe Rejection symptoms: • Oliguria (and progression to anuria?) • Discomfort/pain over allograft • Unexplained flu-like symptoms - nausea, chills, SOB, fever Transplant renal biopsy is performed when an acute or chronic renal allograft rejection is suspected.

Afferent and Efferent Arms of Allograft Rejection

Afferent Arm: • The host becomes sensitized to the donor antigens by means of APCs that present antigens to T cells, the allorecognition process involved 2 different pathways • Direct recognition and indirect recognition Efferent Arm: • Proliferation of alloreactive T cells in lymphoid organs • Direct pathway: - Effector T cells (CD8 T-cell) migrate to blood via thoracic duct and then migrate into the grafted tissue - Graft destroyed by effector T cells • Indirect pathway: - Effector T cells (CD4 T-cells) stimulate antibody production against the graft tissue. - Graft destroyed by antibody induced immune response.

Why are immunosuppressivve drugs given with transplantation?

Although HLA matching significantly improves the success rate of clinical organ transplantation, it does not in itself prevent rejection reactions hence, unless donor and recipient are identical twins, all graft recipients must be given immunosuppressive drugs to prevent rejection!

What is graft rejection? what is it mediated by?

An immunological response mediated primarily by T cells • It is easy to observe and measure a graft rejection in an animal

Alloantigen:

Antigens from another genetically nonidentical member of the same species. Allogeneic.

Xenoantigen:

Antigens from more than one species. Xenogeneic.

Why is HLA matching more critical in HSC tranplant than in solid organ transplantation?

BECAUSE consequences of GVHD are even more aggressive if there is a mismatch of MHC class I or class II

Describe the expression of MHC alleles

Codominant expression

Maintenance Regimes

Currently most transplant heart, lung and kidney transplant centres utilise maintenance regimens consisting of multiple drug therapy using combinations of 2/3 of the following: o Calcineurin inhibitor o An anti-metabolite o A corticosteroid o mTOR inhibitors

Tolerance

Defines the state of specific immunological unresponsiveness of the lymphoid tissue to a given Ag (tolerogen) capable in other circumstances of inducing cell mediated or humoral immunity. Immunological responses to unrelated Ag are not affected by the induction of tolerance as tolerance is specific to the inducing antigen • Central or peripheral tolerance Peripheral tolerance - anergy, etc.

How can bone marrow preparation for HSC transplant in leukaemia patient cause detrimental effects?

Destroying the bone marrow in preparation for HSC transplant can result in immunodeficiency can occur due to depleting the donor HSC transplant of T cells - As the recipient usually has had most of their immune cells destroyed by irradiation and chemotherapy and the donor transplant is lacking T cells - hence the recipient will be lacking T cells and may suffer from an opportunistic infectionIr

Result of Ag recognition in the absence of co-stimulation?

Functional inactivation or deletion (tolerance) • In absence of co-stimulation, a T cell can't respond - can try and induce anergy to promote graft acceptance

Syngeneic:

Genetically similar or identical cells hence immunologically compatible and therefore transplantation does not provoke an immune response

Xenograft:

Graft between different species. Xenogeneic New strategies to help the fact that we don't have enough organ donors

Allograft:

Graft between genetically different individuals of the same species.Allogeneic - This represents the vast majority of transplants

Isograft:

Graft between individuals with identical MHC antigens e.g. identical twins. Syngeneic

Why is patient survival in HLA matched siblings BMT, greater than in HLA matched unrelated donor BMT?

HLA haplotype matching only matches major histocompatibility complexes Minos Has may be different and can stimulate rejection or GVHD • Patient survival is greater in HLA matched siblings than in HLA matched unrelated donors. • HLA haplotype matching only matches major histocompatibility complexes • Minor histocompatibility antigens may be different and can stimulate rejection or GVHD. • Siblings having a higher chance of genetic similarity are more likely to have similar minor histocompatibility antigens to the host than compared to an unrelated donor. • Closer matching will therefore reduce the number of alloantigen's, resulting in a reduced rate of rejection.

Explain the image...

IMAGE - Minor H antigens are peptides derived from polymorphic cellular proteins bound to MHC class I molecules. Self proteins are routinely digested by proteasomes within the cell's cytosol, and peptides derived from them are delivered to the endoplasmic reticulum, where they can bind to MHC class I molecules and be delivered to the cell surface. If a polymorphic protein differs between the graft donor (shown in red on the left) and the recipient (shown in blue on the right), it can give rise to an antigenic peptide (red on the donor cell) that can be recognized by the recipient's T cells as nonself and elicit an immune response. Such antigens are the minor H antigens.

Explain the image...

IMAGE: Top panel: Acute rejection of a kidney graft through the direct pathway of allorecognition. Donor dendritic cells in the graft (in this case a kidney) carry complexes of donor HLA molecules and donor peptides on their surfaces. The dendritic cells are carried via the blood to secondary lymphoid organs (a lymph node is illustrated here), where they move to the T-cell areas. Here, they activate the recipient's T lymphocytes, whose receptors can bind specifically to the complexes of allogeneic donor HLA (both class I and class II) in combination with donor peptides. After activation, the effector T cells travel in the blood to the grafted organ, where they attack cells that display the peptide:HLA molecule complexes for which the T cells are specific.

2 types of immunosuppressive regimens

Induction Regimens • brief use of potent immunosuppressive agents in immediate post-transplant period to reduce the immune response of T cells to the transplanted organ. Individual agents either deplete T cells, or interrupt T cell activation and proliferation • high doses of conventional immunosuppressive agents e.g.: - Corticosteroids, prednisone - Antimetabolites, mycophenolate mofetil or azathioprine - calcineurin inhibitors, cyclosporine or tacrolimus - this can occur really early - often these medications are complemented by using antibody reagents - loading up immune system with lots of different immune interventions • one or more of the following antibody reagents: - IL-2 receptor antagonist, basiliximab - Polyclonal anti-thymocyte antibodies, anti- thymocyte globulin (ATG) - complement can bind to this Ab and cause the T cell to be destoyed - Monoclonal anti-thymocyte antibodies, alemtuzumab • Induction regimes are usually quite severe Maintenance Regimens • Currently most transplant heart, lung and kidney transplant centres utilise maintenance regimens consisting of multiple drug therapy using combinations of 2/3 of the following - calcineurin inhibitor - An anti-metabolite - A corticosteroid • Medications may change throughout the rest of the life of the individual mTOR inhibitors Everolimus - blocks molecules involved in T cell proliferation (but acts after IL-2 has acted on the cell) May have a cocktail of medications which targets IL-2 and other actions • May be blocking of co-stimulatory pathway - makes the T cells anaergic - stops the effector function of T cells - what you want with a transplant

What is currently done for patients undergoing transplantation

MHC matching of recipient and donor + immunosuppression - When you give immunosuppression, you are stopping the T cells from attacking the graft - Without immunosuppression, grafts can be easily rejected

MOA and Adverse effects of azathioprine

MOA: • Antiproliferative agent • antagonizes purine metabolism and may inhibit synthesis of DNA, RNA, and proteins. • May interfere with cellular metabolism and inhibit mitosis. • Mechanism of action is likely due to incorporation of thiopurine analogues into the DNA structure, causing chain termination and cytotoxicity. • Interferes with DNA synthesis - main action on frequently dividing cells • Is a pro-drug - converted in vivo to active form • Cytotoxic drug • Interferes with DNA synthesis main action on frequently dividing cells • Is a pro-drug converted in vivo to active form, which competitively blocks the synthesis of important components of DNA Adverse: Fever, chills, sore throat, body aches, weakness, muscle pain, flu symptoms); severe nausea, vomiting, or diarrhea; pain or burning with urination Accumulation of a drug metabolite in the DNA leads to increased DNA sensitivity to mutations induced by UV rays in sunlight increased risk of skin cancer

MOA and Adverse effects of cyclosporin and tacrolimus

MOA: • CA forms a complex with cyclophilin - complex binds and inhibits calcineurin • Calcineurin inhibitors • (A), tacrolimus (FK506) - inhibit the calcinuerin-dependent activation of NFAT; block IL-2 production by T cells and proliferation by T cells • cyclosporin A and tacrolimus block T-cell proliferation by inhibiting the phosphatase activity of the Ca2+-activated protein phosphatase calcineurin, which is required for the activation of the transcription factor NFAT. • Both drugs reduce the expression of several cytokine genes that are normally induced on T-cell activation, including the gene encoding interleukin (IL)-2, which is an important growth factor for T cells • Cyclosporin A and tacrolimus inhibit T-cell proliferation in response to either specific antigens or allogeneic cells, and are used extensively in medical practice to prevent the rejection of allogeneic organ grafts. • Although the major immunosuppressive effects of both drugs are probably the result of inhibition of T-cell proliferation, they also act on other cells and have a large variety of other immunological effects • Tacrolimus similar to cyclosporin A, but binds to a different immunophilin, but also inhibits calcineurin Cyclosporin A: • Peptide derived from fungus • Forms complex that inhibits calcineurin • Calcineurin is usually required for the activation of cytokine genes normally induced in T cell activation • Overall, decrease T cell proliferation in response to allogenic cells or specific antigens Tacrolimus: • Similar to cyclosporin A except forms complex with the immunophilin molecule called an FK-binding protein • Similarly, this drug:immunophilin complex inhibits calcineurin, and leads to the same downstream effects Adverse: - Cyclosporin - Shaking, headache, dizziness, unusual growth of body hair, nausea/vomiting, diarrhea, stomach upset, or flushing may occur - Affects all immune responses non-selectively - Injures kidney epithelial cells - Tacrolimus - diabetes mellitus, infection, headache, hyperglycemia, hyperkalemia, increased blood urea nitrogen, increased serum creatinine, mental status changes, nephrotoxicity, sensation disorder, and tremor

MOA and Adverse effects of corticosteroids

MOA: • Inhibit inflammation; inhibit ,any targets including cytokine production by macrophages • Act on intracellular receptors • Decrease amount of cytokines and nitric oxide synthase (less NO) • Cross cell membrane bind to intracellular receptors of nuclear receptor family activated glucocorticoid receptors then travel to the nucleus and alter gene transcription • Can effect up to 20% of genes expressed by leukocytes • Immunosuppression via the following anti-inflammatory effects - Decreases amount of cytokines decreased inflammation caused by cytokines - Decreases nitric oxide synthase decreased nitric oxide - Decreased adhesion molecules reduced emigration of leukocytes from vessels Adverse: • Cutaneous effects, electrolyte abnormalities, hypertension, hyperglycemia, pancreatitis, hematologic, immunologic, and neuropsychologic effects (short-term use) - osteoporosis, aseptic joint necrosis, adrenal insufficiency, gastrointestinal, hepatic, and ophthalmologic effects, hyperlipidemia, growth suppression, and possible congenital malformations (long-term use)

What improves the outcome of transplantation? Does this prevent rejection reactions?

Matching donor and recipient at the MHC improves the outcome of transplantation. Although HLA matching significantly improves the success rate of clinical organ transplantation, it does not in itself prevent rejection reactions. - HLA typing/ HLA compatibility testing

Tests to detect alloreactive T cells

Mixed Lymphocyte Reaction (MLR): Detects the presence of alloreactive donor T cells o Lymphocytes from potential donor are mixed with irradiated lymphocytes from the recipient. If naive T cells that recognize alloantigens on the recipient lymphocytes are present, they will proliferate or kill the recipient target cells o Limitation: does not accurately quantify alloreactive T cells. Limiting-dilution assay: which precisely counts the frequency of alloreactive T cells. o A more accurate test is a version of MLR

Does MHC matching guarantee graft survival and why?

NO! In MHC-identical grafts, rejection caused by peptides from other alloantigens bound to graft MHC molecules - Even though MHC genotype might be matched exactly, polymorphism in any other protein could elicit T cell responses that would destroy graft: When donor and recipient are identical at MHC but differ at other genetic loci, graft rejection is not as rapid - The polymorphic antigens responsible for the rejection of MHC-identical grafts are therefore termed minor histocompatibility (H) antigens or minor H antigens. - Can process these and present these to T cells - There are differences in these between the male and female - female may not have immunological tolerance to the antigens as she has not been exposed to them previously - One set of proteins that induce minor H responses is encoded on the male- specific Y chromosome. Female anti- male minor H responses occur

What causes an alloreactive immune response to occur? Where is the alloreactive immune response directed?

Occurs when donor and recipient differ at the MHC. Directed at the non-self allogenic MHC molecule or other molecules on the graft. • Alloantigen = foreign antigen between donor and recipient - The MHC is the foreign molecule - this is the dominant immune response - There are a number of T cells in the body directed against foreign MHC

What two things contribute to MHC diversity?

Polymorphism & Polygeny

Cause of transplant rejection

Primarily due to the strong immune response to non-self MHC molecules on the graft. In most tissues, these will be predominantly MHC class I antigens.

Serological HLA typing

Serological HLA Typing • HLA typing of potential donors and a recipient can be accomplished with a microcytotoxicity test • Leucocytes from potential donors and recipient are distributed into series of wells on microtiter plate • Have a panel of Abs against known HLA molecules - typing/anti-HLA Abs • Ab specific for various class I and class II MHC alleles are added to different wells • After incubation, complement is added to the wells - In presence of Ag and Ab, will bind to Fc part of Ab and lyse the cell - cell will become leaky • cytotoxicity is assessed by the uptake or exclusion of various dyes (e.g., trypan blue or eosin Y) by the cells - if the cell is leaky and dead, it won't be able to exclude the dye (if it was a healthy cell, it would push this dye out) • If the leucocytes express the MHC allele for which a particular Ab is specific, then cells will be lysed upon addition of complement, and these dead cells will take up a dye • HLA typing based on antibody-mediated microcytotoxicity can thus indicate the presence or absence of various MHC alleles IMAGE - Recipient and Donor 1 would match, whereas donor 2 is a mismatch

What does MHC restriction of T cells mean?

T cell specific for peptide x and an MHC molecule produced by a particular MHC allele e.g. MHCa (left panel), will usually not recognize the complex of peptide x bound to a different MHC allele product, MHCb (center panel), or the complex of a different peptide, peptide y, bound to MHCa (right panel). • A particular MHC molecule can bind a particular peptide which is recognised by a particular T cell • A MHC molecule can bind a range of peptides (this is different to the TCR) - as long as the peptide fits in the MHC binding cleft

Frequency of _____ specific for any non-self MHC molecule is relatively high

T cells

Autograft:

The donor is also the recipient. Syngeneic.

The Effector Stage

The immune response to transplanted tissue - involves: - Injury & ischaemia > non-specific inflammatory response - Antigen presentation to T cells increased as expression of adhesion molecules, class II MHC, chemokines, and cytokines is upregulated. - Promotes the shedding of intact, soluble MHC molecules that may activate the indirect allorecognition pathway - T cell cytokines promote macrophage infiltration, endothelial cell activation, more T cells > amplification of response CD8 T cells mediate cytotoxicty > apoptosis - NK cells distinguish allogenic cells from self > potent cytolytic effector mechanisms • once activated, T cells can come into the graft tissue and cause inflammation through the release of cytokines, CD8 T cells recognising graft-specific antigens coming back into the graft, destroying it - CD4 T cells can be helping the CD8 T cells to differentiate, encouraging them to destroy the graft • TNF alpha and TNF beta are made by Th1 cells - are chemotactic and can draw other cells to the site • There is a group of unusual CD4 T cell that are cytotoxic - DON'T WORRY ABOUT THESE • Can get B cells being activated which produce Ab - activate complement and can be involved in ADCC (antibody dependent cytotoxicity)

Self tolerance

Unresponsiveness to self antigens. Forms the basis of our 'normal' inability to mount an immune response to self antigens or endogenous antigens • result of central and peripheral tolerances • tolerance - talking about adaptive immunity

Why is T cell depleted bone marrow used in BMT?

Used to suppress development of GVHD by eliminating mature T cells • Need some T cells, but not too many - can monitor the donor T cells using flow cytometry

Graft versus host disease (GVHD) - what is it? - Main tissues involved? - Characterising features? - Factors present for development - When does it occur? - Ways to prevent

WHAT? Mature donor T cells present in preparations of HSCs and recognize the tissues of the recipient as foreign, causing a severe inflammatory disease in multiple tissues; The converse of graft rejection is graft-versus-host disease (GVHD); One of the major complications of allogeneic HSC transplantation Main tissues involved: skin, intestines, and liver (i.e. mucosal tissues) Characterized by: bright red rash (face, neck, trunk, limbs, palms, soles), diarrhea, liver dysfunction, itch, fever, pneumonitis Factors for development: Recipient must express MHC or minor HC that are lacking in the graft donor Time: Occurs hours to days after the transplant Prevention: Most transplants are therefore undertaken only when the donor and recipient are HLA-matched siblings or, less frequently, when there is an HLA-matched unrelated donor. - Elimination of mature T cells in donor preparation of HSCs in vitro → alloreactive T cells are removed and the immature T cells that mature in the recipient are tolerant to the host self antigens HOWEVER... this can cause immunodeficiency - Immunosuppression very important BECAUSE GVHD mostly occurs in the context of disparities between minor histocompatibility antigens

When are grafts 100% successful?

When grafting from different sites on the same animal or person (autograft), or between genetically identical animals or people (syngeneic graft). Grafts that are syngeneic are permanently accepted

When does graft-versus-host disease develop?

When mature donor T cells present in preparations of HSCs recognize the tissues of the recipient as foreign, causing a severe inflammatory disease in multiple tissues, but particularly involving the skin, intestines, and liver and characterized by rashes, diarrhea, and liver dysfunction . • Because the consequences of GVHD are particularly aggressive when there is mismatch of MHC class I or class II antigens, HLA matching between donor and recipient is more critical than in solid organ transplantation. Graft-versus-host disease is due to donor T cells in the graft that attack the recipient's tissues. After bone marrow transplantation, any mature donor CD4 and CD8 T cells present in the graft that are specific for the recipient's HLA allotypes become activated in secondary lymphoid tissues. Effector CD4 and CD8 T cells move into the circulation and preferentially enter and attack tissues of the graft recipient, particularly epithelial cells of the skin, intestines, and liver that have been damaged by the conditioning regimen of chemotherapy and irradiation prior to transplantation.

First set rejection:

grafts differing at the MHC are rejected around 10-13 days after grafting

HSC Transplant in leukemia - what must happen to recipient bone marrow

must be destroyed by combination of irradiation and chemotherapy

What are some factors that affect graft survival in kidney transplantation?

o ABO blood group identity o Negative cross match o HLA match o Previous blood transfusions o Age, race (graft acceptance may be less likely in an elderly person for example) o Prior disease o Immunosuppressive therapy (even if donors are identical)

Autologous

obtained from the same individual

Allogeneic

relating to or denoting tissues or cells which are genetically dissimilar and hence immunologically incompatible, although from individuals of the same species

Xenogeneic:

relating to or involving tissues or cells belonging to individuals of different species.

Polygeny:

the presence of several different related genes with similar function

Alloimmune Response - initiated by? - Process

• APCs drive T cell activation, therefore drive graft rejection Initiated by dendritic cells - DCs migrating from the graft display peptides from the graft on their surface. - DCs can bring antigens from the graft and their own MHC molecules - the recipient can react to those foreign antigens - the recipient T cells can go back into the grafted organ, resulting in the destruction of that graft - After travelling to lymph node, these APCs encounter naive T cells specific for graft antigens, and stimulate these T cells to divide - The resulting activated effector T cells migrate via the thoracic duct to blood and home to grafted tissue, which they rapidly destroy. - - Can produce cytokines, or if they are a CD8 T cells, can start to destroy the graft cells

Indications for BMT

• Acute lymphoblastic leukemia (ALL) • Acute myelogenous leukemia (AML) • Chronic myelogenous leukemia (CML) • Juvenile myelomonocytic leukemia. • Myelodysplastic syndromes. • Plasma cell disorders. • Hodgkin and non-Hodgkin lymphoma. • Inherited immune disorders (SCID) • Inherited red cell disorders (aplastic anemia) • Bone marrow failure (pathological or medically induced through chemotherapy and radiation therapy) • Autoimmune diseases (experimental)

Post transplant pharmacotherapy induction regimens

• Brief use of potent immunosuppressive agents in immediate post-transplant period to reduce the immune response of T cells to the transplanted organ. Individual agents either deplete T cells, or interrupt T cell activation and proliferation • High doses of conventional immunosuppressive agents e.g.: o Corticosteroids, prednisone o Antimetabolites, mycophenolate mofetil or azathioprine o Calcineurin inhibitors, cyclosporine or tacrolimus • One or more of the following antibody reagent: o IL-2 receptor antagonist, basiliximab o Polyclonal anti-thymocyte antibodies, antithymocyte globulin (ATG) o Monoclonal anti-thymocyte antibodies, alemtuzumab

Clinical features and onset of GVHD

• Can target cells in skin, gut and a variety of other mucosal sites (including the respiratory tract) • Graft contains T cells • Recipient must express MHC or minor HC that are lacking in the graft donor • Clinical - Bright red rash, face, neck trunk, limbs, palms, soles - Itch (histamines can be released by skin mast cells), fever - GIT involvement diarrhoea - Liver function decreases - Lung, BM • Deplete T cells before transplant? - This reduces 'graft versus leukaemia effect', donor T cells may kill residual leukaemic cells • Symptoms generally begin developing after 7-10 post-surgery. Acute GVHD occurs within the first 100 days following a transplant. While chronic GVHD occurs after 100days or more.

role of cyclosporine in controlling rejection

• First drug able to control rejection • Blocks signalling pathways in T cell involved in producing IL-2 - Tacrolimus also acts on this pathway, but in a different way • Originally from a fungus and started to be used in the 1980s - people started to understand the use of HLA typing and immunosuppression - number of graft rejections decreased

Immunosuppressive therapy - uses What happens when there is too little or too much?

• Growth in clinical immunology • Increasing number of diseases are found to be due to aberrant immune responses • Search for drugs capable in inhibiting unwanted immune responses • Nonspecific inhibition of immune responses • Specific immunosuppression • A balance between rejection & infection - Rejection usually due to excessive T cell responses - Too much suppression - may get more infections and cancer - Too much suppression can affect hematopoiesis (bone marrow) - won't be able to fight infectious disease or malignancies • Dampens down the T cell responses in the recipient - T cell won't attack the graft • Important in other immune diseases (e.g. autoimmune - want to dampen down the immune response)

Sequence based typing (SBT) function and procedure

• HLA typing • Determines the nucleotide sequence of an amplified segment of a HLA gene • Accomplished using an automated nucleotide sequencer • The HLA segment is briefly amplified, the excess nucleotides and primers are removed, the amplified DNA is used as a template for a sequencing reaction, and the products of the sequencing reactions are purified and applied to a sequencing gel • Fluorescent labels are used to distinguish chains terminated by each base (A, C, G or T) - Labels are incorporated using a dye-labelled primer or dideoxynucleotide • Do PCR and then read what the sequence is and match it to a data base to tell you what the allele is - so you know exactly what the allele is (because there are 1000s of MHC alleles stored in the database) - This is done through a sequencing reaction containing both normal and modified nucleotides (dideoxynucleotides) which can terminate polymerisation once they are incorporated into the replicating strand of DNA. - A primer is then added to initiate DNA synthesis through DNA polymerase, thus generating primer extension products at every position on the DNA molecule as the polymerisation is terminated at the modified nucleotides. - Fluorescent labels are used to distinguish chains terminated by each base (A, T, G or C) and are incorporated using a dye-labeled primer or dye-labeled dideoxynucleotide terminators. - The primer extension products are separated on a gel that resolves single base differences in DNA and placed into an automated sequencer that uses a laser to excite the dye molecules and records the emissions from each dye. - Software is then used to convert this data into a chromatogram and automate nucleotide assignments for each position. These sequences are then compared to a library of all known sequences to assign the allele/s in the sample.

Second set rejection: when a mouse is grafted for a second time with skin from same donor, it rejects second graft faster. The accelerated response is MHC-specific. - Naive mice that are given T cells from a sensitized donor behave as if they had already been grafted

• If you take a graft from an animal with a particular MHC and put it on another animal with a different MHC, it takes a little bit of time, but the graft is rejected (adaptive immunity) - the cells need to be activated, differentiate, proliferate and make it to the site - First set rejection • Second set rejection - faster rejection because after the first graft, there are many memory cells specific to the graft - proves T cells are responsible for this type of rejection • If you were to take the rejected graft and put it in another animal, it would be rejected quickly also

Groups of complications associated with BMT

• Immune mediated - GVHD and HVGD or failure of engraftment via rejection or insufficient bone marrow cells • Infection - high risk of CMV, and pneumocystis carinii being common causes of death • Cancer - from a hematopoietic origin or due to the radiation and chemotherapy, which are also risk factors for cancer

Complications of kidney transplantation (short and long term)

• Immune suppression to induce host tolerance of solid organ allografts (such as kidney or heart transplants) carries substantial increased risk for infection and malignancy • Urologic and vascular complications may occur. - Urine leak and urinomas - Peritransplant fluid collections - Hematomas - Lymphoceles - Abscesses and infection • Vascular complica- tions include renal artery stenosis and renal artery and renal vein thrombosis. - Infarction - Arteriovenous fistulas and pseudoaneurysms Etc. Short-term: ( infection - UTIs, etc • Blood clots • Acute rejection • Increased BO Long-term: • Cancer • Diabetes • Hypertension Immunodeficiency due to long-term immunnosuppression

Requirements for immunosuppressive agents

• Induction of immunosuppression at the time of initial exposure to the allograft • Maintenance of immunosuppression to prevent allograft rejection • Treatment of rejection episodes - May be able to halt the rejection process • Avoid over-immunosuppression which increases susceptibility to infection and malignancy

Types of immunosuppressants (5)

• Irradiation • Corticosteroids • Cytotoxic agents (target T cells particularly) • Cyclosporin - targets T cells • Antibodies - Use Abs against specific targets in T cell signalling pathway to stop the T cells from being activated • Cyclosporin can affect IL-2 gene transcription (which is T cell growth factor) - the T cells can't proliferate - Need T cells to proliferate in context of infection - Acts across all T cells (not only on MHC-specific T cells) - First drug that was used in transplants • Antibody therapies - Mabs - Antibodies targeting specific molecules (e.g. IL-2 receptor) - blocks receptor so IL-2 can't bind • Other drugs can act more generally on the cell cycle

Reducing risk of infection prior to BMT

• Necessary • Recipients are immunosuppressed - • Prior to BMT, patient undergoes irradiation to kill their lymphocytes • May be perfused with Abs • Management of ventilation systems • Antibiotics • the patient is typically placed on immunosuppressants during the initial period following transplant in order to prevent any donor T cells from promoting GVHD.

High risk of failure in BMT

• Need to destroy all host hematopoietic stem cells prior to transplantation • High risk of HLA mismatch due to HLA polymorphism leading to GVHD • Functional rejection - The risk of failure is high due to large variation in MHC polymorphisms between individuals - A BMT must share some MHC alleles with the host (patient) in order to pass positive selection in the thymus, where thymic epithelial cells display host-MHC. - Failure to share any MHC-alleles with the host means that T-cells cannot be selected and therefore cannot develop, thus functionally BMT is a failure. • Variation in MHC polymorphisms between inidivduals

Why assess the duration of neutropenia in the post-transplant period?

• Neutrophils are the first cells being released in to blood post transplant • Markers of success for BMT • Neutrophils are a marker for engraftment of hematopoietic stem cell transplant • Neutrophils generally appear within 1-2 months of the transplantation. This period is a high risk period for opportunistic infection.

Potential targets for immunomodulation (9)

• Not all of these are currently in use 1. Pretreatment of donor tissue & organs 2. Reduction of histo-incompatibility 3. Inhibition of cytokine production of DC & APC 4. Blocking of Ag recognition 5. Inhibition of cytokine production by TH 6. Inhibition of cytokine activation 7. Inhibition of cell cycle progression 8. Inhibition of clonal expansion 9. Masking target antigens

Xenotransplantation

• Process of grafting or tansplanting organs or tissues between members of different species • Pigs can act as a source of many tissues (e.g. pancreatic islets, neuronal cells, corneas, RBCs, etc. • Tissues need to be engineered to avoid human immune response • The availability of these mutlitansgenic pigs together with the novel immunosuppressive and anti-inflammatory agents is likely to overcome any remaining pathobiological problems that currently prevent xenotransplantation from being introduced successfully into clinical practice There is a high risk of rejection Get rid of MHC locus from pig and replace it with human MHC and the pig generates human MHC - less likely to be rejected

What is cross matching?

• Recipient serum potentially containing donor-specific anti-HLA antibodies is added to donor T or B lymphocytes (because they can express MHC molecules), along with complement (A). - If the Abs find something on the donor cells to bind to, they will bind and complement will bind to the Fc part of the Ab and cause them to be destroyed (as complement causes lysis) • If donor-specific antibodies are not present, no lysis occurs and the result is deemed negative (B). • If donor-specific anti-HLA antibodies bind to the lymphocytes and then activate complement, cell lysis will occur and the crossmatch result will be deemed positive (C). - Won't use this donor for the recipient • Same principle as microcytotoxicity assay, but different use - looking for Abs in patient's serum

What are the signs and histology of kidney graft rejection?

• Signs - Flu-like symptoms - Fever - Decreased urine output - Weight gain - Pain or tenderness over transplant - Fatigue - Increasing serum creatinine lvevel - Decreased urine production - Organ function decreases • Histology - Most severe changes are in small arteries, arterioles and veins - Vessels: early changes are neutrophil margination in peritubular interstitial capillaries, swelling and vacuolization of endothelial cells with ulceration, chronic inflammatory cells in intima and vacuolization of smooth muscle cells in media; thrombi are often small and non-occlusive, but in irreversible rejection, become obliterative and widespread with necrosis of vessel wall - Glomeruli: endothelial cell swelling, increased cellularity and occasional thrombosis - Tubules: focal necrosis - Interstitium: haemorrhage - Lots of lymphocytes - acute - Only few neutrophils - Tubule surrounded by inflammatory cells - possibly undergoing early necrosis

What is the point of tissue typing and what ways can it be carried out?

• Tissue typing = HLA typing - examine HLA of patient and potential donor to see how well they match - 6 antigen matiching (2 x HLA-A, -B, -DR) ϖ there are six identified antigens that have an important role in transplantation - Microcytotoxicity assay ϖ Utilises serum with known anti-HLA anitbodies that recognise particular HLA loci in order to match genetically similar individuals in hope of performing a tissue transplantation ϖ a complement-dependent lymph cytotoxicity assay. Frozen typing trays containing wells coated with different antibodies against HLA antigens are commercially available ϖ The HLA type of an individual is determined by interpretation of the reaction of the individual's lymphocytes with different antisera used in the complement- dependent lymph cytotoxicity test. ϖ see how well the HLA matches ϖ there are 6 possible HLA genes to match - HLA-A, HLA-B and HLA-DR should match ϖ Each gene has one allele from the mother and one from the father - Microlymphocytotoxicity Assay ϖ complement-mediated serologic assay in which peripheral blood lymphocytes are first mixed with an antiserum containing specific anti-HLA antibodies. ϖ After a period of incubation, complement is added and the mixture is further incubated ϖ If the lymphocytes carry the HLA antigen against which the antibosied in the antiserum are directed, an antigen-antibody reaction will occur, which activates the complement ϖ The ensuing enzymatic cascade culminates in membrane damage that ultimately kills the lymphocytes ϖ A positive reaction is ascertained by examining the cell suspension for cell death - this can be accomplished in many ways including phase contrast microscopy coupled with use of supra vital stains 1. Peripheral blood lymphocytes (PBLs) are isolated by Ficoli-Hypaque centrifugation and added to a panel of HLA typing sera (with each containing alloantibodies to specific HLA antigens). 2. If the antibodies bind the antigens on the cell surface, it will activate serum complement and cause cell death, yielding a positive reaction. If there is no binding of antibodies, there will be no reaction, yielding a negative reaction. 3. Each test well is scored as a percentage of dead cells and the overall reaction pattern of the typing sera is interpreted to give the HLA antigen phenotype of the individual.

Progenitor cell sources used for BMT:

• Umbilical cord blood • Bone marrow from donor siblings • Peripheral blood

Progenitor cell sources for BMT

• Umbilical cord blood • Haploidentical donor • Mismatched unrelated donor Previously harvested and stored PBSCs or donor progenitor cells - inject GM-CSF to mobilise stem cells in peripheral blood

What are the requirements and considerations for kidney transplantation?

• Very successful - the success rate has increased • Absolute requirements for the allocation of donor kidneys include: o ABO identity or compatibility o Negative T cell cross match (don't want the recipient's plasma to have Abs that can react with the donor MHC) o No previous Ab against donor HLA Ag o No shared incompatibilities with previous donor(s) • Other considerations: o Degree of HLA match (still can transplant organs that don't have a 100% match because it is done with immunosuppressive therapy) o CMV status o Blood transfusion Need to screen donor tissue for infection

Warm and cold ischaemic times

• Warm ischaemic time - amount of time that an organ remains at body temperature after its blood supply has been stopped or reduced • Cold ischaemic time - amount of time that an organ is chilled or cold and not receiving a blood supply - in general, the sooner an organ can be transplanted, the better - begins when organs put into cold solution after surgery and ends after the tissue reaches physiological temperature • Want to reduce these times - graft acceptance is better the shorter these times are

What are the 3 main types of compatibility testing/matching?

• Way to try and prevent graft rejection • ABO compatibility - E.g. Blood type O can only have O donor • Tissue Typing (HLA typing): o Trying to get the recipient and donor as close as possible with their MHC o Direct or indirect recognition o HLA type of the patient and the potential donor to see how well they match o 6 antigen matching, 2x HLA- A, -B, -DR o E.g. A1, A2/B8, B51/DR17, DR11 o Microcytotoxicty assay, molecular methods, MLR (mixed lymphocyte reaction - looks for differences between donor and recipient lymphocytes) • Cross matching: o Presence of antibodies to HLA = HLA "sensitised"or other donor Ag o HLA antibody levels can change following events such as blood transfusions, previous transplants, pregnancies, miscarriages, even surgeries o Checking there are no Abs in the recipient's plasma/serum that will react with the donor molecules o Important in preventing hyperacute reaction ϖ The Abs in the recipient may bind to vascular endothelium in the graft, initiating the complement and clotting cascades - blood vessels in the graft become obstructed by clots and leak, causing haemorrhage of blood into the graft - Also do cross-matching when doing blood donations • May also do a serology test - Do a viral serology on donor prior to transplant to look for HIV, etc. to make sure you don't give it to the recipient

Graft-versus leukemia effect

• Where the donor T cells in the allogeneic preparations of HSCs recognised minor histocompatibility antigens expressed by the leukemic cells and kill the leukemic cells • therapeutic effects of HSC transplantation in leukemia • Donor BM and T cells recognise the leukemia Ags as being foreign - having a few T cells coming through in the transplant will help eliminate leukemia - but don't want too many as it will cause GVHD

Transplant rejection images

• early attempts to transplant tissues failed • rejection of transplanted tissue was associated with inflammation and lymphocyte infiltration - there are many nuclei - lots of inflammatory cells (a lot of them are lymphocytes, being mononuclear) - damage graft cells LEFT IMAGE = NORMAL RIGHT IMAGE = TRANSPLANTED TISSUE

what are the different types of organs transplanted routinely in medicine?

• graft survival varies depending on the type of organ it is - kidneys are pretty well accepted • people are looking at the different signals (1,2 and 3) and how they can be altered to minimise graft rejection