Lecture 4 Flashcards by Molly Fowler

How do we enrich for antibodies that recognise the CD4 binding site of HIV, gp120?

create a probe that mimics the CD4 binding site (RSC3)
create a variant that has a single amino acid change within the CD4 binding site (Delta RSC3)
Select for antibodies that bind the probe (b12), but not the variant – i.e. retain CD4 binding-site recognition
antibodies that bind both (2G12) are not specific for CD4 binding site
use RSC3 and delta RSC3 to isolate B cells (specific markers sorted via FACS) from peripheral blood of HIV patients that encode CD4-binding site specific antibodies

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What are markers for B cells ?

CD20 and CD19 are only expressed on B cells

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What does the expression of IgG mean for a B cell?

It means they have been through an immune response
they’ve received T cell help
they’ve proliferated
probably been into a GC
undergone CSR

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What is the method for large-scale isolation of bNt Mabs from Memory B cells?

Sort single IgG B cells that bind a modified gp160 trimer called 2CC
Recover the IgV genes from each cell
Re-express as a recombinant, complete IgG in a test tube

you have a probe that is part of the HIV virus
you find B cells which are isotype switched and that recognise this prove
isolate them
recover their V genes, from each individual cell, Heavy and Light chain V region genes
Re-express them in another cell type in a test tube
can make that cell now produce that antibody
so you’ve gone from a circulating B cell in the blood of a patient to an Ab factory in your laboratory that is making an anti-HIV antibody that may have the properties of neutralisation because it binds to a site that you think is important to that process
you can also get info about that V region genes for example the number of somatic mutations they have

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What are some more strategies to isolate and characterise HIV-specific antibodies from HIV-infected individuals?

Firstly you screen their serum to see if they have the desired Abs or not. (binding assay)

take WBCs –> high throughput sequencing - don’t try to isolate anything just whack the whole stuff through a HTS machine and see which Ig sequences come back most often – represent the B cells mounting a response at the time you have isolated the blood. Compare them in an attempt to work out what the mutational history is and what they came from.
Try to identify the individual rare B cells that are recognising the virus by using parts of the virus as a bait to fish them out. If you do it through this process of cell sorting you can attach other attributes e.g. it has to be a B cell, isotype switched etc - enrich for the kind of cell that you are interested in
alternatively you can take everything - recover the V genes from these cells, remake the antibody and re-express it in a cell type in a laboratory dish - you can then analyse what it looks like, what it binds to and does it work in neutralisation

you’ve then gone from a patient with serum Abs to actually purifying those Abs that have desirable properties

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What is VRC01? What has been done with it?

an antibody that blanks out completely the ability of HIV to bind to CD4 and so prevents infection
highly mutated V gene
idea was that if we could find out what it recognised at the beginning before it went into the GC we’d know what we had to immunise people with in order to trigger that response and guide it through the process of affinity maturation to make these good high-affinity Abs
Took the V gene sequences and identify all the somatic mutations and change them back to what they were in the very beginning at the onset of the immune response
it actually no longer bound to HIV
only acquired the capacity to bind to HIV by virtue of these mutations
turns out this is not uncommon
if so, then how did they get to react to the virus in the first place? if in these sorts of assays they don’t recognise it
turns out the assay is wrong rather than the immunology - tested the wrong form of the virus against their capacity to bind

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How do response, escape and counter-response shape the Ab repertoire to HIV?

antibodies acquire increasing numbers of SHM in GC
rapid excape mutation in Env
Increasing breadth and potency of neutralisation
successive emergence of broadly neutralising Abs in patient
First Abs are strain specific
Then V2 specific bNAbs to emerge
Followed by a 3rd wave to as yet unknown epitope
possible late bNAbs do not recognise initiating virus
Each HIV escape is into an increasingly limited ‘space’
important to remember that the virus has parts of it that can change - and it does - these also happens to be the bits that your immune system most likes to recognise, other parts of the virus are unchangeable - change would mean that it lost its capacity to infect - this is the most difficult to make a response to and usually happens much later

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How can we rationally design Anti-HIV Mabs with increased and improved neutralising activity?

can one improve Ab binding to gp120 by changing individual amino acids?
examine the binding site at the amino acid level of Vh and CD4
make an AA exchange to make Vh more like CD4 - Swap G for Phe?
Swapping Vh G for an aromatic amino acid (Y, W, F) greatly improves affinity - 10 fold improvement by a single aa change

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What has injecting mice with broadly neutralising Mabs show us?

mice transplanted with human CD34+ stem cells - humanised
this reconstitutes the human immune system in the mice (B, T macrophage etc)
mice infected with HIV1 generate stable viraemia for > 60d (i.e. HIV replication model)
Test if neutralising Mabs provide long term protection
Mono-therapy with bnMabs is ineffective –> 5 different Mabs and 1 HIV strain, neither on their own effective
tri-therapy is partly effective - injecting three different Mabs gave some mice protective levels
Penta-therapy with bnMabs is effective - most/all mice showed controlled, low levels of HIV-1 when treated with all 5 Mabs
But there is a viral rebound as Abs disappear - as they slip below a particular level due to not being constitutively made the virus is eventually able to start replicating once more - this means that the Mabs are not curative, merely protective - once below a certain threshold the virus is able to escape from that suppression and start infecting things

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What is important about showing that penta-therapy with broadly neutralising Mabs is effective?

Shows that it is possible to contain HIV with sufficient range of neutralising specificities, which might inform vaccine design
reveals a therapeutic approach to suppress HIV-1 viraemia, possibly helping those who are unable to take HART or in conjunction with HART
Possible for long-term production of Abs in people using Adenovirus vectors
Also shows what is required for prophylaxis and a way of stopping vertical transmission

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What are the (original 6) hallmarks of cancer?

sustaining proliferation
evading growth suppression
metastasis - dissemination
immortality of replication - senescence
blood supply / inducing angiogenesis
blocking apoptosis (cell death)

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How does a cell become a cancer cell?

a cancer is the uncontrolled growth of a cell
unrestrained in its proliferation and in its location
its those properties of expanding number and distribution which ultimately cause pathology and death
cells have a lot of inbuilt mechanisms to prevent cancer from developing and each of these have to be overcome for a normal cell to be transformed into a cancerous cell
these properties that a cell has to acquire are not normal - they require a change in the normal expression pattern either by enforced expression of a gene which shouldn’t be expressed or by mutations in a gene to change its properties so it can no longer be regulated or it gains additional features
mutation is a key driver of cancer

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What must the molecules that promote B-cell cancer do?

promote cell division
extend cell life span by enhancing survival
enable DNA mutation
metastasis not necessary for these circulating cells
special properties of B cels that can predispose them to malignancy

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How are B cells in GCs similar to cancer cells?

they are proliferating like crazy
not responsive to signals which stop their growth
able to circulate throughout the body
have processes to prevent their death and accumulate DNA damage while doing that
i.e. proliferating B cells in germinal centres are already a reasonable distance along the path to becoming a cancer cell

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How do you find translocations?

cytogenetic techniques where you look for banding patterns - low resolution
last 30/40 years people have developed probes that label whole stretches of chromosome, you can ‘paint’ the chromosome, can see using colour the translocations. could combine this with a probe for an oncogene e.g. Myc - a very potent growth promoting gene (older - 3/4 years ago but still widely used in clinic)
more sophisticated and global assessment is to actually just sequence everything
- sequencing the genome has actually become a relatively trivial thing, can be done for less than $1000, in only a couple of hours - a day at the most
possible to take tumours from people, to sequence them, and to sequence all of the chromosomes.
if you have little tiny fragments and you sequence them from both ends, and then map those sequences back onto the genome you can find in some cases that instead of just being a continuous sequence (e.g. myc or heavy chain locus) it actually swaps from one to the other
in this way, at a nucleotide level, you can actually see that there is a translocation by comparing the tumour DNA with the DNA of a healthy cell - exact
this helps to produce a map of recurrent chromosomal translocations in B-cell cancers

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What are the risks of being a B cell?

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immunoglobulin class switch recombination (a chromosomal rearrangement) and somatic hypermutation
designed to enhance antibody affinity and diversity, and provide memory
but with a cost: most B cell lymphomas originate from germinal centre B cells, as indicated by somatically mutated Ig genes and chromosomal translocations involving the Ig locus
double stranded breaks of a chromosome are clearly a precursor to a translocation, changing the context of a gene and therefore changing its regulation
AID has off target effects, it’s not exclusively restricted to the process of SHM, so those mutations may change the properties of some of the genes which ordinarily shouldn’t be behaving in a particular way/gives them a new property

To where do these common translocations go?

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Ig loci
B cells make immunoglobulin, constitutively
by translocating these oncogenes into these loci, it ensures that the genes are the other end of the translocation are going to continue to be expressed by the B cell
B cell may want to turn them off, but because they are now linked up with the Ig loci, it can’t

What is the role of Activation Induced Cytidine Deaminase (AID) in germinal centres?

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IN GC, B-cells undergo multiple rounds of proliferation, suppressing differentiation
AID expression is restricted to proliferating B cells in the GC (dark zone)
i.e. rapidly dividing cells expressing an enzyme that is designed to alter their DNA
These cells are also expressing another molecule called BCL-6 – absolutely required for GC to form but it has a lot of very dangerous properties: it permits the GC reaction to proceed, permits DNA damage to occur - relatively risky in terms of accumulating damage
AID triggers events that promote chromosomal rearrangement and gene mutation

How do you find translocations?

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cytogenetic techniques where you look for banding patterns

What translocation is common in follicular lymphoma?

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Very frequently translocations of anti-apoptosis gene BCL2 into Ig locus

What are some genes commonly translocated and so deregulated in B cell cancers?

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Myc, cyclins: cell cycle and growth regulators; promotes cell division
Bcl2, Bclx: cell survival proteins; extends life span
Bcl6/p53: DNA damage sensors; increases genomic instability/mutation
(Bcl6 suppresses p53 which is a DNA damage sensor)
Ordinarily Bcl6 is only activated transiently to allow for the rearrangement of Ig genes however constitutive expression means the cell is not able to check for DNA damage that is out of the ordinary –> cancerous

What translocation is common in Burkitt lymphoma?

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Myc into the heavy chain locus

What is Myc?

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Transcription factor with wide influence (global regulator of the genes necessary for cell growth)

drives proliferation: up-regulates cyclins, down-regulates cyclin inhibitors
regulates cell growth: enhances ribosomal RNA and protein synthesis
inhibits differentiation
Myc is a very strong proto-oncogene, found to be abnormally up-regulated in many types of cancers (e.g. Burkitts Lymphoma)

What translocation is common in follicular lymphoma?

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Very frequently translocations of anti-apoptosis gene BCL2 into Ig locus

What are growth regulators?

Cell cycle regulators Typically resting cells are in G0 and need to be triggered to enter into the cell cycle done by acting cyclins and associated kinases. Get cells out of G0 into G1 and the S phase. drivers: cyclins are transiently expressed, they activate CDKs (cyclin-dependent kinases) and drive the cell cycle brakes: several inhibitors (p21, p15, p18 etc) bind to specific CDKs and inhibit their activity The inhibitors block cell cycle progression, often to allow time to repair DNA damage (p53 is a DNA damage sensor)

What signals stimulate a cell to undergo programmed cell death?

Apoptosis can be triggered by: - Anoikis: loss of contact/being put out of location - Cytokine withdrawal: removal of sustaining growth factors - Glucocorticoids: mediate stress - DNA damage

In what human B cell lymphomas does over-expression of Bcl-2 often have a role?

Particularly common in: - Follicular (almost always carries with it a translocation of Bcl-2 into the Ig locus: as long as that cell tries to express Ig, it will express large quantities of Bcl-2, effectively presenting this cell from committing suicide, despite damage to its DNA, movement in the wrong location, loss of growth factors, etc) - CLL Around 50% of cases in Large cell Rare in Hodgkin and Burkitt

What is Bcl6?

- Bcl6 expression is absolutely required for GC to form Bcl6 is a transcriptional repressor - normal function and regulation: - - inhibits B cell differentiation during the germinal centre reaction to enable isotype switching and affinity maturation - - blocks the DNA damage response (by blocking a number of genes) and cell cycle regulation (represses p53 and the DNA damage sensor ATR) - Translocation and mutation - common in DLBCL - - causes aberrant Bcl6 expression so DNA damage occurs unchecked, while also inhibiting differentiation

What happens when the cell is under stress?

- cells activate BH3-only - an excess of these proteins overwhelm Bcl2 and trigger apoptosis by perforating mitochondria, releasing Cyt C (represents the end of the road) and ultimately, activation of caspases (chew up everything inside the cell)

What would happen if you had an excess of Bcl-2?

- would be very difficult for the cell in which that occurred to trigger death in response to any of the regular danger/damage signals - no matter what they do with the BH3-only proteins, there is always enough of the Bcl-2 to prevent the activation of apoptosis - over-expression of Bcl2 blocks apoptosis (cell death) - Bcl2 is a very weak oncogene since by itself it cannot promote cell proliferation - blocking apoptosis, however, is very common in cancer

In what human B cell lymphomas does over-expression of Bcl-2 often have a role?

What are some mutations that convert Bcl6 into an oncogene?

- Translocations that lead to continued expression - Somatic mutations that stop transcription from being repressed - AID - also mutations that stop Bcl6 protein from being degraded - increased stability

What are the roles of Bcl6 in normal GC B cells?

- allow proliferation in the presence of DNA damage - suppress differentiation - suppress apoptosis - blocks activation

Where do signals that switch off BCL6 come from?

- CD40 and BCR

What happens once BCL6 is switched off?

- Activation, selection and differentiation of cells expressing high affinity Ig receptors - Elimination of cells damaged or expressing low affinity Ig receptors

What are some mutations that convert Bcl6 into an oncogene?

- Translocations that lead to continued expression - Somatic mutations that stop transcription from being repressed - also mutations that stop Bcl6 protein from being degraded - increased stability

What is IRF4?

A repressor of Bcl6 expression

Lecture 4 Flashcards

(37 cards)