L20,L19,L18,L16 forgotten Flashcards
L20- Cancer therapies?
Co-stimulatory receptor agonist antibodies target the co-stimulatory (‘accelerators’) receptors (signal 2) to positively (+) regulate T cell activation and responses
T cell regulation depends on a balance of positive (co-stimulatory receptors) and negative (immune checkpoints) regulators of T cell activation. Clinical development has focused on the immune checkpoints. However, the positive regulators of T cell activation (diverse co-stimulatory receptors) are also rational targets for cancer immunotherapy. Antibody drugs (agonists) targeting these receptors have shown promise in the early stages of clinical testing (e.g. antibodies: anti-CD28, ICOS, OX40, CD27).
Disadvantage: these immune agonists may induce strong non-specific immune activation (cytokine release syndrome/auto-immunity risk). Have to be tested cautiously.
ICB immunotherapy: Immune checkpoint blockade (ICB) immunotherapy (including anti-CTLA-4, anti-PD-1 and anti-PD-L1 antibody drugs) which target and block immune checkpoint molecules can unleash anti-tumor immune responses (T cell activation and effector functions) and has provided a new weapon against cancer.
ICB in the clinic –
Harnessing TME cells to treat cancer
ICB antibodies (anti-CTLA-4, anti-PD-1 and anti-PD-L1) can unleash anti-tumor immunity (T cell activation and effector functions) and mediate durable clinical responses in subsets of patients
Cd8 and cd26 same ligands for cd28- costimulatory receptor so antibodies do not target them for therapy but ctla-4 directly.
Anti-PD-1 antibody ICB immunotherapy (with nivolumab) has substantial clinical activity against melanoma and lymphoma patients.
T cell-mediated lysis (tumour kill)
ICB therapy induces T cell recognition
and lytic immune synapse formation
Treating leukaemia (CLL) and lymphoma (DLBCL) tumor cells with anti-PD-L1 or T cells with PD-1 prevents tumour inhibitory signalling and rescues anti-tumor T cell activity.
Mechanisms of PD-L1 pathway-induced immunosuppression in the tumour microenvironment
t cell apoptosis, t cell exhaustion, treg induction.
L20- PD-1 characteristics?
PD-1 is absent on resting naïve and memory T cells and is expressed upon TCR engagement.
In contrast to CTLA-4, PD-1 expression on the surface of activated T cells requires transcriptional activation, and thus is delayed (6–12 hr after activation).
PD-1 engagement can also activate the inhibitory phosphatase PP2A. PD-1 engagement directly inhibits TCR-mediated effector functions.
`PD-1 has important physiologic role in restraining collateral tissue damage during T cell responses to infection. Prevent t cells from being overactivated.
Immune checkpoint receptors (PD-1, CTLA-4) and their ligands (PD-L1/2) are upregulated in cancer
PD-1 ligands (PD-L1, PD-L2) are upregulated in cancer
PD-1 receptor is highly expressed on T cells from cancer patients (TILs) – chronic stimulation by tumour antigens (Ags) drives “T cell exhaustion”
Co-option of the immune checkpoint network (ligands and receptors) is a central process by which tumours resist elimination by endogenous tumor-specific T cells
In healthy tissues- pdl-1 normally expressed at relatively low levels on dendritic cells?
So increased expression= more pd-1 engaged with the ligand. Interacts much more readily. Ligands can be expressed by tumour themselves or by dendritic cells. Drives immune suppression as if t cell interacts with cancer=suppressed more readily by cancer cells.
Mechanisms for why the ligands are overexpressed in cancer: cancer cells have oncogenic protein pathways which are activated e.g: akt. When tumour cell has oncogenic signalling, pd-L1 is expressed at very higher levels. Called innate resistance or intrinsic resistance:
Intrinsic resistance refers to the constitutive expression of PD-1 ligands because of genetic alterations or activation of oncogenic pathways in cancer cells.
Constitutive expression of PD-1 ligands - amplification of chromosome 9p24.1 in Hodgkin’s lymphoma which encodes PD-L1, PD-L2 and JAK2.
EBV viral infection is another mechanism of PD-L1 overexpression in the lymphomas.
Alternativley: adaptive resistance. T cell recognises signal 1 and secrete ifn-y which is taken up by tumour cell, triggers stats signalling that elavates pd-L1.
Adaptive resistance refers to the induction of tumour PD-L1 expression in response to cytokine IFN-γ secreted by proximal T cells.
Adaptation of tumor cells upon sensing an inflammatory immune/T cell response.
Inhibition of T cells also mediated by PD-L1+ myeloid cells or DCs in the tumour microenvironment
L19- Live, attenuated or inactivated/killed
bacterial and viral vaccines?
Effective vaccines are composed of intact microbes that are treated in such
a way that they are attenuated OR killed, so they no longer cause disease,
while retaining their immunogenicity
Advantage of attenuated microbial vaccines is that they elicit all the innate and
adaptive immune responses (both humoral and T cell mediated)
However, the inactivated (killed) bacterial vaccines generally induce limited
protection and are effective for only short periods
Live, attenuated viral vaccines are usually more effective e.g. polio, measles,
and yellow fever.
Early approach for producing attenuated viruses was repeated passage in cell
culture
More recently, temperature-sensitive and gene deletion mutants have been
generated
Viral vaccines often induce long-lasting specific immunity, so immunisation of
children is sufficient for lifelong protection
So good cus they activate innate and adaptive-memory b and t cells produced. The innactibated killed are not as potent, limited protection, do not have long lasting effects.
Learn examples of pathogens associated with vacines
Attenuated: formed in lab through repeat passage in cell culture.
In live-attenuated
vaccines, like the
measles, mumps, and
rubella shot, weakened
viruses incorporate their
genetic instructions into
host cells, causing the
body to churn out viral
copies that elicit
antibody and CD4 and
CD8 T cell response
a
The major concern with attenuated viral or bacterial vaccines is safety
The live-attenuated oral polio vaccine has nearly eradicated the disease, but in
rare cases the virus in the vaccine is reactivated and itself causes serious polio
Success of worldwide vaccination is creating the unusual problem that the
vaccine-induced disease, although rare, could become more frequent than the
naturally acquired disease! So vacine can be infectous.
Solution? reverting to the killed virus vaccine to complete the eradication
program (or 3rd generation vaccines)
A widely used inactivated vaccine of considerable public health importance is
the influenza or flu vaccine. Influenza viruses are grown in chicken eggs
Innactivated vaccine: Flu shot: Trivalent inactivated (killed) vaccine given intramuscularly
3 of the most frequently encountered influenza strains are selected every year
and incorporated into this vaccine
L19- Viral vectors - live viral vaccines – recombinant viruses?
Gene based approac (3rd generation) viral vector is source of antigen. Infect cells and induce t cell response.
Alternative gene based approach - introduce genes
encoding microbial antigens into a non-cytopathic virus and to
infect individuals with this virus
Thus, the virus serves as a source of the antigen
The great advantage of viral vectors is that they, like other
live viruses, induce the full complement of immune
responses, including strong CD8+ T cell/CTL responses
This technique uses vaccinia virus vectors, and canarypox
viral vectors, which are not pathogenic in humans
Recombinant viruses induces both humoral and T cell-
mediated immunity against the antigen produced by the
foreign gene
A potential problem - the recombinant viruses may infect host
cells, although they are not pathogenic, they may produce
antigens that stimulate CTL responses that kill the infected
host cells. These and other safety concerns have limited
widespread use of viral vectors for vaccine delivery
Gene-based vaccines carry the genetic
instructions for the host’s cells to make the
antigen, which more closely mimics a natural
infection
The viral vector technique transports genetic
information in a less harmful virus — often a
common cold–causing adenovirus —
sometimes engineered so it can’t replicate in
the host
ChAdOx1nCoV-19 a non–replicating viral
vector candidate in phase 3 trials from
AstraZeneca and the University of Oxford,
uses an adenovirus that infects chimpanzees
instead of humans. But, it’s possible that
cross-reacting pre-existing immunity to human
adenoviruses could still diminish the
response.
L19- VariZIG
VariZIG (Varicella Zoster Immune Globulin (Human)) is a sterile lyophilised preparation of purified human immunoglobulin G (IgG) containing antibodies to varicella zoster virus, VZV (chickenpox). It provides passive immunisation for non-immune individuals exposed to VZV, reducing the severity of varicella infections
L19- pathogens associated with vaccines?
FIRST GENERATION: Live attenuated or killed bacteria- cholera, bacillus calmette -guerin
FIRST GENERATION: Live attenuated or killed viruses- polio, influenza, rabies, yellow fever, measles
A widely used inactivated vaccine of considerable public health importance is the influenza or flu vaccine. Influenza viruses are grown in chicken eggs
Flu shot: Trivalent inactivated (killed) vaccine given intramuscularly
3 of the most frequently encountered influenza strains are selected every year and incorporated into this vaccine
SECOND GENERATION: Subunit antigen vaccines: tetanus toxoid, diphtheria toxoid
SECOND GENERATION: conjugate vaccines: haemophilius influenza, pneumoccocus
SECOND GENERATION: synthetic antigens: hepatitis (recombiant proteins)
THESE ARE ALL PROTEIN BASED VACCINES
THIRD GENERATION GENE BASED VACCINES:
viral vectors: HIV antigens in canarypox vector
DNA vaccines/ mRNA
A widely used inactivated vaccine of considerable public health importance is the influenza or flu vaccine. Influenza viruses are grown in chicken eggs
Flu shot: Trivalent inactivated (killed) vaccine given intramuscularly
3 of the most frequently encountered influenza strains are selected every year and incorporated into this vaccine
L16- Anabolism regulators?
If cells are in good environment with glucose, oxygen and growth factors (gf firstly activate pI3K and then mTOR. mTOR is activated and promotes Hif1-a (alpha) and c-myc: 2 transcription factors. C-myc- tf that tells cells to synthesise proteins and therefore anabolism to gen elements and divide and grow. Synthesise membranes, dna and proteins. biosynthesis of proteins and lipids drives cell cycle and cell cycle progression.
L16- when does catabolism>anabolism?
Ratio between amp and atp changes (amp greater than atp). Amp kinase blocks mTOR and hif1-alpha as cell does not have enough glucose to synthesise things. Happens in tumour microenvironment. Ampk activation is a problem but is a natural way for cell to control atp prod. energy conservation programme: glucose to OXPHOS. blockade of protein and lipid biosynthesis.
L16- GAPDH FUNCTION?
Primary (metabolic) role: In glycolysis, GAPDH converts glyceraldehyde-3-phosphate to 1,3-bisphosphoglycerate, a critical step in energy production.
Moonlighting (non-metabolic) role: In resting T cells, GAPDH binds to interferon-gamma (IFN-γ) mRNA and blocks its translation by preventing the ribosome from attaching to the mRNA.
L13- what are T cell characteristics and how do they migrate through SLO? + CTLA-4
- Part of the adaptive immune response.
- Express CD3 as part of their antigen recognition receptor (they are T cells)
and CD4 (they are T-helper) in their surface. - Orchestrators of the adaptive immune response. 3 main functions:
- Secrete cytokines.
- Interact with B cells to produce antibodies.
- Regulate the immune response.
Signal 1 without signal 2 = anergy
Signal 3: cytokines drive differentiation
Upon correct signalling from a DC, CD4+
T cells activate: - Proliferation
- Survival
- Metabolic reprogramming (IM lecture)
- Differentiation
Migration through SLOs is controlled by chemokines, L-selectin
and integrins.
Circulating lymphocyte enters a high endothelial venule in lymph node
Binding of L-selectin (CD62L) on t cell to GlyCAM-1 and CD34 ( ligands on endothelial cells) allows rolling interaction
LFA-1 (integrin on t cells) is activated by CCR7 (a chemokine receptor on t cells) signalling in response to CCL21 or CCL19 (chemokines bound to endothelial surface)
Activated LFA-1 binds tightly to ICAM-1
(anchoring)
Lymphocyte crosses the endothelium and enters lymph node via diapedesis
The ability of CTLA-4 to inhibit T cell responses under conditions of low B7 expression is crucial for maintaining immune homeostasis and preventing overactivation, especially in scenarios like chronic inflammation or autoimmunity.
However, it can also be exploited by tumors to suppress anti-tumor immunity, which is why CTLA-4 inhibitors (e.g., ipilimumab) are used in cancer immunotherapy.
This is dependent on level of antigen
Clonal contraction: 90-95% effector
cells die. The surviving cells will form
the memory population
L11- RAG
The joining mechanism for recombining gene segments during V(D)J recombination is initiated by the enzymes RAG1 and RAG2 (Recombination Activating Genes 1 and 2). These proteins recognize recombination signal sequences (RSS) flanking the V, D, and J gene segments and bring the segments together to facilitate recombination.
This process involves:
Formation of hairpin loops:
The DNA is cut at the RSS, and the free ends of the DNA form hairpin loops to maintain genome integrity (to avoid open free DNA).
Opening the hairpins:
The hairpins are then opened at random points by enzymes, introducing junctional diversity. This imprecision at the site of joining results in diversity in the final recombined gene sequence.
Adding palindromic (P) nucleotides:
When the hairpins are opened, the DNA ends often form overhangs, creating a palindromic sequence (P nucleotides) as they fold back on themselves.
Adding or removing nucleotides:
To create a blunt end for joining, nucleotides may be removed from the overhangs, or additional random nucleotides (N nucleotides) are inserted into the gap by the enzyme Terminal deoxynucleotidyl transferase (TdT). N nucleotide addition is entirely random and significantly enhances diversity.
Imprecise joining:
The imprecise nature of this process—where hairpins are opened, and nucleotides are added or removed—creates further variability and is a key contributor to junctional diversity.
Combinatorial diversity:
This mechanism works alongside the random combination of V, D, and J segments (combinatorial diversity) to generate a vast repertoire of antibodies or T cell receptors.
easier explanation:
1. What is V(D)J recombination?
This is a process where B cells and T cells create a huge variety of antibodies (for B cells) or T-cell receptors (for T cells) to recognize many different pathogens.
It achieves this by rearranging specific segments of DNA called V (Variable), D (Diversity), and J (Joining) segments.
2. Role of RAG1 and RAG2
RAG1 and RAG2 are special enzymes that cut DNA at specific places called Recombination Signal Sequences (RSS) next to the V, D, and J segments.
These enzymes bring one V segment, one D segment, and one J segment close together for recombination.
3. Formation of Hairpin Loops
After RAG enzymes cut the DNA, the free ends of the DNA fold into hairpin loops.
Why? This helps protect the DNA from breaking apart completely.
4. Opening the Hairpins
Another enzyme comes in and opens the hairpins at random spots.
This is important because the randomness creates diversity in the resulting DNA sequence.
5. Adding Palindromic (P) Nucleotides
When the hairpins are opened, the ends of the DNA sometimes form small sequences that read the same forward and backward (like “GAAG” or “CAGC”).
These are called P nucleotides and are created naturally as the DNA folds back on itself.
6. Adding or Removing Extra Nucleotides
An enzyme called Terminal deoxynucleotidyl transferase (TdT) can:
Add random nucleotides (N nucleotides) to the open ends.
Remove existing nucleotides if needed to make the DNA ends blunt.
This randomness is called junctional diversity and is another way the immune system generates variety.
7. Joining the DNA
The DNA ends are then joined together to form a complete V-D-J segment.
The joining isn’t perfect—it’s imprecise, but this imprecision adds even more variety to the final antibody or receptor.
8. Combinatorial Diversity
On top of the randomness of junctional diversity, the immune system can mix and match different V, D, and J segments to create an enormous number of combinations.
L11- Number of genes + CDR formation?
Rearrangement of immunoglobulin genes: germline configuration- immature b cells. Dont need to know exact number of genes but magnitude. 50 v genes 30 d 6 j Being selected and recombined with the d segment and intervening segments are lost. But crucially the bits upstream of that are still there. Only bits in between are lost from the genome.
Within v segments: framework regions making the physical immunoglobulin structures and within thema re CDRs- the unstructured loops at the ends and 2 of them are encoded by the v segments. Within 3rd cdr= formed by that junction of vdj being brought together.
Junction between VDJ sequence determines antigen specificity: the cd3 is important in determining what the final outcome is going to be.
L11- BM microenvironment?
stromal cells line the bone marrow
- mixture of endothelial, fibroblasts, fat cells
- provide growth signals to developing B cells
* Provide essential cell-cell contact
-VLA-4/VCAM-1 (CAMs)
- Kit/SCF
* AND soluble growth factors (cytokines)
- Interleukin 7
Differentiation of B cells in the bone marrow from HSCs:
Bone marrow microenvironment and cell-cell contact initiate the recombination process. IL-7 further drives the recombination process. Developing B cells are exposed to these signals in the bone marrow, and they control the processes involved in generating diversity, such as the rearrangement of the immunoglobulin genes
L10- granulysin?
activating receptors?
What happens when activating or inhibitory receptors are activated?
NK function
Initial synapse purpose
granulysin- has antimicrobial actions and can induce apoptosis.
- KIR receptors (activating) work through ADCC. NKG2D- C-type lectin MIC-A AND MIC-B ON TUMOUR AND VIRALLY INFECTED CELLS
-ITAM AND ITIMS- DOCK PHOSPHATASES THAT DEPHOSPHORYLATE LIPIDS AND PROTEINS PRODUCED FROM DOWNSTREAM SIGNALLING
- NK FUNCTION: ADCC,
NK functions
* kill virus-infected target cells and tumour cells
* produce and release immunoregulatory cytokines (IFN-gamma, TNF-alpha,
GM-CSF and chemokines) that activate macrophages to destroy
phagocytosed microbes (innate NK cells can influence other innate
immune cells – macrophages)
Produce IFN-Y increases the capacity of
macrophages to kill
phagocytosed bacteria, similar
to IFN-γ produced by T cells.
This allows time for T cell–
mediated immunity to develop
and eradicate the infection. so takes time as immediate response by nk and macrophages but adaptive response which is antigen specific takes several days to develop.
INITIAL SYNAPSE:
Naive CD8+ T cells circulate and upon encountering antigen by a naive t cell presented by professional APCs, these naive T cells slow down—engages the dendritic cell via integrin signaling— and T cell receptor (TCR) activation triggers the formation of the initial signaling “immune synapse” always use the word initial.
This initial ‘signaling’ immune synapse induces remarkable clonal expansion (this effector differentiation occurs over the next 4–5 days)
-> naive CD8+ T cells differentiate into heavily ‘armed’ effector CD8+ T cells or also
known as CTLs that are loaded with specialized cytolytic granules that contain
perforin and granzymes
Once effector CD8+ CTLs recognise their target cells in the periphery, a
“lytic immune synapse” is formed, and the cytolytic granules are secreted.
