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4.1 Transfusion and Transplant Immunology > Practical 3: Immunohistochemistry for Rejection > Flashcards

Practical 3: Immunohistochemistry for Rejection Flashcards

1
Q

What is the renal corpuscle and what does it do?

A

Its the Bowman’s capsule and the glomerulus

It filters plasma from capillaries into the renal tubules

Glomerulus at the centre (a meshwork of capillaries), surrounded by the Bowman’s capsule, the space in between is known as the Bowman’s space

This is where the ultra-filtrate of plasma is first collected

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2
Q

Talk about CD45 as a rejection marker, what is it, were is it found

A

CD45 is a transmembrane glycoprotein

Its a protein tyrosine phosphatase receptor type C

It was originally calle lucocyte common antigen (LCA)

The gene is located on chromosome 1 in humans

It can activate SRC tyrosine kinases e.g. p56Lck

Spice variants result in various isoforms

CD45RA is expressed by naive T cells while CD45RO is experessed by activated and memory T cells

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3
Q

Talk about CD68 as a marker

A

It is a transmembrane glycoprotein highly expressed by cells of the monocyte lineage e.g. monocytes, osteoclasts and macrophages

Scavenger receptor found on monocytes in blood and macrophages in tissue

Gene is located on chromosome 17 in humans

A scavenger receptor binds

Binds oxLDL, phosphatidylserine on apoptotic cells

Mainly located on the endosomal/lysosomal membrane but can rapidly translocate to surface membrane

May have a negative effect on MHC presentation of exogenous antigen

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4
Q

Talk about CD19

A

A transmembrane glycoprotein
Gene located on chromosome 16
Expressed on all B cell stages except plasma cells
Functions as an adaptor protein that recreuits cytoplasmic signalling proteins and reduces the threshold for signal transduction from the BCR for B cell activation

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5
Q

Talk about CD20

A

A B cell marker, a non-glycosylated phosphoprotein member of the MS4A family which forms a tetraspan membrane-bound protein
Encoded by a gene on chromosome 11
The extracellular portion provided the docking site for anti-CD20 mAbs binding
CD200 is expressed on the surface of all B cells beginning at the pro-B phase and progressively increasing in concentration until maturity
It is thought to funtion as a modulator of cell growth and differentiation, and to initiate intracellular signals

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6
Q

Talk about CD3

A

Expressed only by T cells
Always associated with the TCR-produces the TCR complex
Responsible for signal transduction
Composed of 2 heterodimers and, in90% of human and murine T cells, a homodimer of zeta chain

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7
Q

What are some signs of acute rejection in the kidney

A

Neutrophil infiltration of the glomerulus and interstitium - also seen in ischamic injury

Lymphocytic infiltrate of the renal tubules (not so much the glomeruli)

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8
Q

What is the Banff Classification of Allograft Pathology?

A

A standardised working classification system developed in 1991 which has contributed to the standardisation of definitions for histologic injuries resulting from renal allograft rejections and provided a universal grading system for assessing these injuries

It is used to clarify the mechanism of renal allograft injury, thus providing a guide to clinical therapy

Its how pathologists report to transplantation surgeons on rejection

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9
Q

What is the Banff Classification based on?

A

It dinstinguishes between immune and non-immune injury as well as classified the type of rejection as antibody-mediated rejection (AMR) or T cell mediated rejection (TCMR), both of which may be acute or chronic based on specific pathological findings

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10
Q

What IHC marker does the Banff classification use?

A

It uses C4d as a marker of renal pathology associated with active AMR

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11
Q

What are PTCs?

A

Peritubular capillaries - tiny blood vessel in the kidney

They surround the cortical parts of the proximal and distal tubules -> not the same as the vasa recta which goes into the medulla to approach the loop of Henle i.e. PTCs are capillaries of the cortex while vasa recta are capillaries of the medulla

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12
Q

Explain how a Banff score is assigned

A

Look at histology of peritubular capillaries - scored according to the number of inflammatory cells present within the capillary lumina

Level of C4d staining in the same tissue also contributes to the score

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13
Q

What exactly is C4d?

A

C4d is the end product of C4 regulation by factor 1

C4 -> C4b -> iC4b (by factor 1) -> C4d (by factor 1)

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14
Q

What is AMR, how does it occur, how does it affect the tissue

A

Acute Antibody Mediated Rejection

Abs against donor Ags bind to Ags expressed on endothelial cells in the graft vessesl
The subsequent compplement activation and cell adhesion results in endothelial cell necrosis, followed by platelet deposition and coagulation
Mononuclear cells adhere to the endothelium of the glomeruli and the peritubular capillaries
This process is accompanied by C4d deposition in both tissues

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15
Q

How do we use C4d to investigate AMR

A

C4d negative staining in peritubular capillaries seen in early development of antibody-mediated rejection

CD45 showing glomerular and peritubular infiltration 2 days post transplant = rejection - treatment needed

Persistant peritubulat capillaritis with Cd4+ cells - 12 days post-transplant

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16
Q

What are some histologic signs of chronic rejection

A

Intimal damage to blood vessels
Less obvious mononuclear cell infiltrate
Progressive deposition of material in the interstitial space (IS)
Thickening of the IS which causes damage to the tubules, blood vessels and glomeruli

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17
Q

c4d positive

A

Marker of the activation of the classical pathway of the comlpement system

Diagnosis of AMR by 4d depotision in PTCs

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18
Q

CD3, CD4, CD8 positivity

A

CD3 in T cells
CD4 in helper T cells
CD8 in cytotoxic T cells

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19
Q

CD68 positivity

A

Positive in monocytes and tissue macrophages

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20
Q

CD68 positivity

A

Positive in monocytes and tissue macrophages

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21
Q

CD56 positive

A

Positive in NK cells and cytototoxic T cells

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22
Q

CD25 positive

A

Receptor for interleukin 2 and a maker of T cell activation

To identify the activated T cells in TCMR

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23
Q

MIB-1 positive

A

A nuclear protein related to cell proliferation or cycle

To examine the proliferation of T cells (used with CD3 marker)

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24
Q

PCNA positive

A

Proliferating cell nuclear antigen

marker of proliferation

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25
Q

Perforin positive

A

A glycoprotein for pore formation in cell membranes of target cells

Positive in NK cells and cytotoxic T cells

used to identify cytotoxic T cells in TCMR

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26
Q

Granzyme B positive

A

Expressed in cytoplasmic granules of activated cytotoxic T cells and NKs

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27
Q

TIA-1 positive

A

A cytotoxic granule-associated protein expressed in NK cells and cytotoxic T cells

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28
Q

CD38/CD138 (syndecan-1)

A

Positive in plasma cells

To detect plasma cell-rich acute rejection

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29
Q

CD20/CD79a

A

Positive in B cells
To detect posttransplantation lymphoproliferative disorder

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30
Q

Myeloperoxidase (MPO)

A

Positive in polymorphonuclear leukocytes
To clairfy the type of infiltrating cells in allograft rejection

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31
Q

FOXP3

A

Positive in Treg cells
(Controversial findings in allograft rejection)

32
Q

IL-17

A

Positive in Th17 cells
The expression of IL-17 increases in allograft rejection

33
Q

alphaSMA (aSMA)

A

Positive in vascular smooth muscle cells and interstitial myofibroblasts

Used to examine the thickening of the initma in chronic rejection or the degree of interstitial fibrosis

34
Q

type IV collagen

A

Positive in glomerular and tubular basement membrane

35
Q

CD31

A

Positive in endothelial cells

To identify and evaluate the PTC and glomerular capillaries

36
Q

D2-40

A

Positive in lymphatic endothelium
To distinguish lymph vessels from PTC

37
Q

Give a general note on immunosuppression in transplantation

A

A majority of patients receive organs mismatched at one or more HLA loci
To prevent rejection, immunosuppressant drugs are administered
These drugs leave patients highly susceptible to infection especially when at greatests levels pre and post transplantation
The dose of drugs is gradually reduced to maintenance levels which prevent acute rejection while sustaining active defennces against infection - likelihood for chronic rejection increases
Each drug also has various side effects so they are often used in combination to increase immunosuppressive effects but not toxic effects
There is a x3 times higher risk of malignant disease when on these drugs for long periods of time e.g carcinomas, lymphomas, kaposi’s sarcoma

38
Q

What are the three classes of immunosuprresive drugs?

A

Non-specific depletion of the majority of lymphocytes and monocytes and the inhibition of the responsiveness of the reamining few e.g. Abs and corticosteroids given prior to transplantation

Interference with 3 signals needed for T cell activation

Cytotoxic drugs which have their affect after naive T cells have begun to proliferate

39
Q

Give three examples of interfering with 3 signals needed for T cell activation

A

The signal from the TCR (TCR/CD3) - NFkB and NFAT

The costimulatory signal (B7/CD28)

The signal generated by IL-2 binding to its receptor

40
Q

What are class 1 immunosuppressive drugs, give two examples?

A

Antibodies that react broadly with WBCS
They are given prior to and post transplantation to deplete these cells
- rabbit antithymocyte globulin
- Alemtuzumab (rat mIgG ant-CD52)

41
Q

What is rabbit antithymocyte globulin?

A

A polyclonal mixture of high affinity antibodies that binds T, B, NK, dendriticand endothelial clls, fixes complement and acts as opsonins

42
Q

Wht is Alemtuzumab?

A

Humanised rat mIgG that is specific for CD52

CD52 is expressed on almost all lymphocytes, monocytes and macrophages

Therefore it results in a profound, long-kasting lymphopenia

43
Q

How does Alemtuzumab work?

A

The cell surface complex of CD52 and anti-CD52 (alemtuzumab) is unusually efficient at fixing complement -> complement mediated cell lysis

Antibodies that bind CD52 are very close to the cell membrane increasing the likelihood that C3b will bind covalently to the leukocte suface

44
Q

What are corticosteroids, give two examples?

A

Drugs such as hydrocortisone (cortisol) and prednisone

Drugs which do not act on cell-surface receptors but instead diffuse across the plasma membrane and bind cytoplasmic receptor

They are most effective when administered prior to transplantation

They alone insufficiently immunosupress to prevent graft rejction but work well in combination with cytotoxic drugs

45
Q

What is hydrocortisone (cortisol)?

A

A steroid made by the adrenal crotex

It has been made clinically for more than 50 years to reduce inflammation

46
Q

What is prednisone?

A

A synthetic derivative of hydrocortisone, it is a pro-drug

When enzymatically converted into prednisolone it is about 4 times more potent in reducing inflammation

47
Q

How do corticosteroids immunosuppress?

A

The prevent the action of NFkB by increasing the production of IkBa (inhibitor)

48
Q

Explain the pathway of how steroids work

A

In the cytosol of the cell, steroid receptors form complexes with heat shock protein Hsp30

Steroids diffuse across the cell membrane and bins to this complex, releasing Hsp30

The steriod receptor now crosses the nuclear membrane and enters the nucleus

Here is binds to gene regulatory sequences and activates transcription where they increase the production of IkBa (inhibitor)

49
Q

What does IkB normally do, how is this affected by steroids?

A

IkB normally binds NF-KB keeping it in the cytosol

steroids increase IKB production thus keeping NF-KB in the cytosol, preventing gene regulation e.g. IL2 production

50
Q

How is NFKB normally activated

A

DAG activates PKCtheta which leadds to the assembly of a membrane-bund coplex
- CARMA1, BCL-10, MALT1

This complex activate a multiprotein enzyme complex (IKKa, IKKB, IKKy), inhibitor of KB kinase (IKK)

IKK phosphorylates the inhibitor of IKB

IKB now releases NF-KB allowing it to move into the nuclease

51
Q

What are some of the affects of corticosteroid therapy?

A

Decrease in inflammatory cytokine production e.g. IL1, TNF-alpha etc
Decrease in adhesion moleules
Prednisolone alters lymphpocyte homing, lymphocytes are barred from entering the SLT, instead the congregate in the BM
Induction of apoptosis in lymphocytes and eosinophils

52
Q

What are some of the side effects of corticosteroid therapy?

A

Fluid retention
Weight gain
Daibetes
Loss of bone mineral
Thinning of skin

53
Q

Talk about blocking signals from the TCR-NFAT activation by calcineurin, give two examples

A

Introduced in the 1970s - used throughout 80s and 90s (cyclosporin era of drugs)

Cyclosporin A
Tacrolimus

54
Q

How does cyclosporin A wor

A

Its a cyclic decapeptide derived from the soil fungus Tolypocladium inflatum

It inhibits T cell activation by disrupting transduction signals from the TCR

Cyosporin A binds to cyclophilin which binds calcineurin preventing its activation by Ca2+ and thus blocking nFAT activation

55
Q

How does Tacrolimus work

A

Tacrolimus is isolated from a soil actinomycete

Its a macrolide, structurally distinct from cyclosporin A but works in similar way

Tacrolimus binds FK-binding protein which then binds clacineurin, preventing its activation by Ca2+ and thus blocking NFAT activation

56
Q

What are some side effects of Cyclosporin and Tacrolimus

A

Since these drugs dont effect proliferatin cells, haematopoiesis and intestinal damage do not occur

Nephrotoxicity may occur in patients who become sensitised, these can no longer tolerate the drug

57
Q

What affects do tacrlimus and cyclosporin A have

A

The reduce expression of T cytokines such as IL-2 and TNF-a

reduced cell division of T cells (lack of IL2)

No cytokines to act on B cells -> cells cant divide, cells will apoptose once activated without these cytokines

58
Q

Give an example of an antibody specific for CD3

A

Introduced in 1986, CD3-specific mouse mAB OKT3

59
Q

How is OKT3 used?
(3)

A

Treatment involved a 5-15 day course of daily injections of mouse-CD3 antibodis combined with prednisolone

Anti-CD3 caused the TCRs to be internalised and therefore they were unable to recognise antigens

Attempts to convert therapeutic mouse anti-CD3 antibodies into either chimeric or humanised forms were unsuccessful - hence taken off market

60
Q

What was OKT3 replaced with?

A

Anti-CD52 and rATG

61
Q

Talk about the use of a combination of immunosuppressants to prevent rejection

A

After transplantation patients are maintained on a combination of immunouppressive drugs that because of their cytotoxicity and the immunodeficiency they cause, are gradually reduced in dosage

As dosage is increased the risk of chronic rejection increases

Sometimes early symotoms of rejection appear

62
Q

Why was OKT3 taken of the market?

A

Its an anti human CD3 mouse mAb but we were unable to humanise it/make a monoclonal antibody

It ended up causing serum sickness, an extreme form of type III hypersensitivity which causes skin haemorrhages and an urticarial rash

Immune complexes are deposited in small blood vessels - activates complement and phagocytosis - induces fever

Immune reaction to the mouse antigens

63
Q

Give four examples of drugs which block signals

A

Belatocept
Basiliximab
Daclizumab
Rapamycin (sirolimus)

64
Q

What is Belatocept?

A

A soluble, synthetic chimeric protein that combines the extracellular domain of CTLA-4 with the hings and fc domains of the IgG1 heavy chain

Acts to prevent alloreactive T cells receiving their co-stimulatory signal from activated DCs expressing B7 and presenting alloantigen

It works as well as the older drugs but preserves kidney function more, it is associated with increased incidence of episodes of acute rejection

65
Q

How does IL-2 act normally?

A

IL-2 acts in both an autocrine and paracrine fashion by binding to the IL-2 receptor expressed by T cells

Naive alloreactive T cells express a low-affinity receptor for IL-2 (consists of B and y chains)

On recognition of alloag the alpha chain (CD25) is synthesised - high affinity receptor

66
Q

What are two IL-2 signal blocking drugs?

A

Chimeric basiliximab
Humanised daclizumab

Both are IgG mAbs specific for CD25

67
Q

How do basiliximab and daclizumab work?

A

They are both mAbs specific for CD25

Thes are first given just before the transplant is performed

Subsequent infusions are gien during the first 2 months after transplantation

Benefit is that this treatment targets T cells embarking on activation - does not induce significant immunodeficiency

68
Q

How does basiliximab and daclizumab only work on partially activated T cells

A

Anti-CD25 anitbodies dont bind to the low affinity IL-2 receptor on naive alloreactive T cells

Anti-CD25 will bind to the high affinity IL2 receptor on activated T cells and prevents the generation of signal 3

69
Q

What are cytotoxic drugs?

A

These kill alloantigen-activated proliferatin T cells -> hence they are given after transplantation

70
Q

List the cytotoxic drugs

A

Azathioprine
Mycophenalate mofetil
Cyclophosphamide
Methotrexate

71
Q

What is azathioprine

A

A pro-drug much used in kidney transplantation, administered after transplantation

Its eventually converted to 6-thioinosinic acid which inhibits the production of inosinic acid, an intermediate in the production of adenine and guanne - this inhibits DNA replication

Like all cytotoxic drugs it damages all tissues normally active in cell division e.g. bone marrow, intestinal epithelium, hair follcles etc

Same side effects as chemo: anaemia, leukopenia, thromboytopenia, intestinal damage, hair loss, hepatotoxic

72
Q

What is mycophenalate mofetil?

A

A product of penicillium stoloniferum
Its metabolise in the liver to mycophenolic acid
It inhibits inosine monophosphate dehydrogenase, necessary for guanine synthesis

73
Q

What is cyclophosphamide?

A

N2 mustard compound developed as a chemical weapon
Used heavily during WWI
Its a pro-drug converted to phosphoramide mustard which alkylates and cross-links DNA molecules
This affects normal cell division and transcription
Equally immunosuppressive pre and post antigen stimulation
In addition to side-effects shared with other cytotoxic drugs it specifically damages the bladder (cancer or haemorrhagic cystitis)
Not particularly toxic to the liver - useful alternative -used in short courses

74
Q

What is methotrexate?

A

A drug found to be very effective in cancer treatment

It prevents DNA replication by inhibiting dihydrofolate reductase which is essential for the synthesis of thymidine (thymine + deoxyribose)

Its the drug of chouce for inhibiting GVHD in bone marrow transplant receipients

75
Q

Sirolimus

A

Binds FK-binding protein like Tacrolimus but prevents T cell activation by inhibiting signal transduction from the IL-2R

It also induces Treg development

It impedes progression through the proliferation cycle by inhibiting p70 S6 kinase and activity of cdk2-cyclin E complex

76
Q

Sirolimus

A

In notes

4.1 Transfusion and Transplant Immunology (24 decks)

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