Practical 3: Immunohistochemistry for Rejection Flashcards
What is the renal corpuscle and what does it do?
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
Talk about CD45 as a rejection marker, what is it, were is it found
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
Talk about CD68 as a marker
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
Talk about CD19
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
Talk about CD20
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
Talk about CD3
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
What are some signs of acute rejection in the kidney
Neutrophil infiltration of the glomerulus and interstitium - also seen in ischamic injury
Lymphocytic infiltrate of the renal tubules (not so much the glomeruli)
What is the Banff Classification of Allograft Pathology?
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
What is the Banff Classification based on?
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
What IHC marker does the Banff classification use?
It uses C4d as a marker of renal pathology associated with active AMR
What are PTCs?
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
Explain how a Banff score is assigned
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
What exactly is C4d?
C4d is the end product of C4 regulation by factor 1
C4 -> C4b -> iC4b (by factor 1) -> C4d (by factor 1)
What is AMR, how does it occur, how does it affect the tissue
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
How do we use C4d to investigate AMR
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
What are some histologic signs of chronic rejection
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
c4d positive
Marker of the activation of the classical pathway of the comlpement system
Diagnosis of AMR by 4d depotision in PTCs
CD3, CD4, CD8 positivity
CD3 in T cells
CD4 in helper T cells
CD8 in cytotoxic T cells
CD68 positivity
Positive in monocytes and tissue macrophages
CD68 positivity
Positive in monocytes and tissue macrophages
CD56 positive
Positive in NK cells and cytototoxic T cells
CD25 positive
Receptor for interleukin 2 and a maker of T cell activation
To identify the activated T cells in TCMR
MIB-1 positive
A nuclear protein related to cell proliferation or cycle
To examine the proliferation of T cells (used with CD3 marker)
PCNA positive
Proliferating cell nuclear antigen
marker of proliferation
Perforin positive
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
Granzyme B positive
Expressed in cytoplasmic granules of activated cytotoxic T cells and NKs
TIA-1 positive
A cytotoxic granule-associated protein expressed in NK cells and cytotoxic T cells
CD38/CD138 (syndecan-1)
Positive in plasma cells
To detect plasma cell-rich acute rejection
CD20/CD79a
Positive in B cells
To detect posttransplantation lymphoproliferative disorder
Myeloperoxidase (MPO)
Positive in polymorphonuclear leukocytes
To clairfy the type of infiltrating cells in allograft rejection
FOXP3
Positive in Treg cells
(Controversial findings in allograft rejection)
IL-17
Positive in Th17 cells
The expression of IL-17 increases in allograft rejection
alphaSMA (aSMA)
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
type IV collagen
Positive in glomerular and tubular basement membrane
CD31
Positive in endothelial cells
To identify and evaluate the PTC and glomerular capillaries
D2-40
Positive in lymphatic endothelium
To distinguish lymph vessels from PTC
Give a general note on immunosuppression in transplantation
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
What are the three classes of immunosuprresive drugs?
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
Give three examples of interfering with 3 signals needed for T cell activation
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
What are class 1 immunosuppressive drugs, give two examples?
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)
What is rabbit antithymocyte globulin?
A polyclonal mixture of high affinity antibodies that binds T, B, NK, dendriticand endothelial clls, fixes complement and acts as opsonins
Wht is Alemtuzumab?
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
How does Alemtuzumab work?
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
What are corticosteroids, give two examples?
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
What is hydrocortisone (cortisol)?
A steroid made by the adrenal crotex
It has been made clinically for more than 50 years to reduce inflammation
What is prednisone?
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
How do corticosteroids immunosuppress?
The prevent the action of NFkB by increasing the production of IkBa (inhibitor)
Explain the pathway of how steroids work
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)
What does IkB normally do, how is this affected by steroids?
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
How is NFKB normally activated
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
What are some of the affects of corticosteroid therapy?
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
What are some of the side effects of corticosteroid therapy?
Fluid retention
Weight gain
Daibetes
Loss of bone mineral
Thinning of skin
Talk about blocking signals from the TCR-NFAT activation by calcineurin, give two examples
Introduced in the 1970s - used throughout 80s and 90s (cyclosporin era of drugs)
Cyclosporin A
Tacrolimus
How does cyclosporin A wor
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
How does Tacrolimus work
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
What are some side effects of Cyclosporin and Tacrolimus
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
What affects do tacrlimus and cyclosporin A have
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
Give an example of an antibody specific for CD3
Introduced in 1986, CD3-specific mouse mAB OKT3
How is OKT3 used?
(3)
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
What was OKT3 replaced with?
Anti-CD52 and rATG
Talk about the use of a combination of immunosuppressants to prevent rejection
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
Why was OKT3 taken of the market?
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
Give four examples of drugs which block signals
Belatocept
Basiliximab
Daclizumab
Rapamycin (sirolimus)
What is Belatocept?
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
How does IL-2 act normally?
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
What are two IL-2 signal blocking drugs?
Chimeric basiliximab
Humanised daclizumab
Both are IgG mAbs specific for CD25
How do basiliximab and daclizumab work?
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
How does basiliximab and daclizumab only work on partially activated T cells
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
What are cytotoxic drugs?
These kill alloantigen-activated proliferatin T cells -> hence they are given after transplantation
List the cytotoxic drugs
Azathioprine
Mycophenalate mofetil
Cyclophosphamide
Methotrexate
What is azathioprine
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
What is mycophenalate mofetil?
A product of penicillium stoloniferum
Its metabolise in the liver to mycophenolic acid
It inhibits inosine monophosphate dehydrogenase, necessary for guanine synthesis
What is cyclophosphamide?
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
What is methotrexate?
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
Sirolimus
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
Sirolimus
In notes
