Week 9 Flashcards
For innate immunity,
- Define innate immunity.
- What types of cells are involved?
- What immunue mediators are involved?
- Innate immunity
- First responder to infection, tissue damage, and inflammation
- Cells involved: include neutrophils, dendritic cells, macrophages, natural killer cells
- Immune mediators involved: interferons, cytokines, and chemokines
For adaptive immunity,
- Define adaptive immunity.
- What does it require?
- What cells are involved?
- What are the two types?
- Highly specific recognition of antigens
- Requires clonal expansion of memory cells
- Self-reactivity leading to autoimmune disease
- Cells involved: B-cells, T-cells, dendritic cells, and macrophages
- Types
- Humoral
- Cell-Mediated
What are the cells and their functions in:
- humoral immunity
- cell-mediated immunity
- Humoral
- B cells – creates antibodies (memory cell)
- Block infections and eliminate extracellular microbes
- Cell-mediated
- Cytotoxic T-cells – kills infected cells and eliminate reservoirs of infection (memory cell)
- Helper T-cells – eliminates phagocytosed microbes (memory cell)
What are the three major cell types?
- Lymphocytes
- Antigen-presenting cells
- Effector Cells
What two types of cells are called lymphocytes and how do they progress through their life cycle?
- Lymphocytes
- B-cells: humoral immunity
- T-cells: cell-mediated immunity
- Life Cycle: naïve cell → effector lymphocyte → memory lymphocyte
What cells types are in the subtype: antigen-presenting cells?
- What is the function of each cell type?
- Dendritic cells: initiation of T-cell response
- Macrophages: effector phase of cell-mediated immunity
- Follicular dendritic cells: initiation of B-cell response
What cells types are in the subtype: effector cells?
- What is the function of each cell type?
- T-cells: activation of phagocytes and killing of infected cells
- Macrophages: phagocytosis and killing of microbes
- Granulocytes: kills microbes
What are the four postulates in “The Clonal Selection Hypothesis”?
- Clonal Selection Hypothesis
- Each lymphocyte bears a single type of receptor with a unique specificity (by V(D)J recombination)
- Virus must bind to lymphocyte receptor for lymphocyte activation (selection)
- Daughter lymphocyte cells derived from an activated lymphocyte will bear receptors of identical specificity as the parental cell (clonal)
- Lymphocytes bearing receptors for endogenous molecules will be deleted at an early stage
Differentiate between primary and secondary immune responses.
- In primary immune response, response is slow because naïve B-cells must undergo V(D)J recombination in order to respond to first contact with an antigen → creates memory B-cells
- In secondary immune response, memory B-cells can respond quicker because of acquired specificity to antigen
Where do B-Cells and T-Cells mature?
- B-cells maturate in bone marrow
- T-cells maturate in thymus
What are some secondary homes for B-Cells and T-Cells?
- Within these tissues, where are B-cells and T-cells located?
- Secondary homes: B-cells peripherally, T-cells centrally
- Spleen
- Lymph nodes
- GALTs – gut-associated lymphoid tissues
- MALTs – mucosal-associated lymphoid tissues
What are the five phases of immune response in situ?
- “First responders” = innate immune cells
- Antigen presentation to naïve adaptive immune cells
- Clonal expansion; amplification of antibody and cytokines
- Pathogen elimination; contraction phase of the immune response
- Memory
What kind of cells can act as antigen presenting cells (APCs)?
- Antigens are captured by dendritic cells, macrophages, and B-Cells (pAPCs) in the epithelium
What is the process of antigen presentation leading to T-Cell
- Antigens are captured in the epithelium.
- Blood borne antigens are captured in the spleen
- Tissue resident APCs migrate to lymph nodes
- T-Cell activation
For Cytotoxic T-Cells, what is:
- the surface marker?
- the function?
- location of antigen source within a cell?
- MHC Class recognized?
- Surface Marker: CD8
- Function: Cell Killing
- Antigen Source: Cytosolic
- MHC Recognized: MHC Class I
For Helper T-Cells, what is:
- the surface marker?
- the function?
- location of antigen source within a cell?
- MHC Class recognized?
- Surface Marker: CD4
- Function: Promote macrophage activity and B-Cell function
- Antigen Source: endosomal
- MHC Recognized: MHC Class II
What is the structure of MHC Class I?
- How many alpha and beta chains?
- Expressed on what ells?
- Binds peptides of what size?
- MHC Class I
- 3 alpha chains and 1 beta chain
- Expressed on all somatic cells
- Binds short peptides (8 to 11 AAs)
What is the structure of MHC Class II?
- How many alpha and beta chains?
- Expressed on what ells?
- Binds peptides of what size?
- 2 alpha chains and 2 beta chains
- Expressed only on professional APCs (dendritic, B, and macrophage)
- Binds long peptides (11 to 30 AAs)
What facilitates the MHC binding proteins to bind peptides?
- How MHC Proteins bind peptides
- Binding site of MHC molecule only requires 2 to 3 AAs (anchor peptides)
- MHC molecules can bind large repertoire of peptides
What are the three important characterisitics of MHC proteins?
- MHC genes are polygenic (multiple genes code for single phenotype)
- MHC genes are polymorphic (high number of mutations)
- MHC gene expression is codominant (mom and dad alleles of MHC class proteins are equally expressed → 6 class I molecules can be expressed)
What are the three functions of the polymorphic nature of MHC genes?
- MHC genes are polymorphic (high number of mutations)
- Allows for diversity of peptides to bind
- Alters contact with T Cell Receptor
- Each T Cell Receptor can only recognize a single peptide/MHC combo
What are the three functions of the invariant chain?
- Binds newly formed MHCII in ER
- Blocks peptide binding groove
- Targets complex to endosome (Ii is degraded in endosome → CLIP (peptide of invariant chain) is unloaded from MHCII by HLA-DM)
In which pathway can cross presentation occur (MHC I or MHC II)?
- Professional APCs can activate both MHC I and II presenting pathways without being infected
- Normally, MHC I pathway happens in infected cells
What causes Type I Bare Lymphocyte Syndrome?
- Type I – loss of MHC class I expression
- Mutations in TAP
What causes Type II Bare Lymphocyte Syndrome?
- Type II – loss of MHC class II expression
- Reduced number of T helper cells
- Much more detrimental
What causes Type III Bare Lymphocyte Syndrome?
- Type III – loss of both I and II
- Similar to type II
What viruses inhbiit the Class I MHC pathway and at what step?
- Viruses that inhibit MHC Class I pathway
- Herpes simplex – blocks peptide binding to TAP
- Adenovirus – retention of class I in the ER
- Cytomegalovirus – targets class I to cytosol
What pathogens escape the MHC Class II pathway and therefore require cytotoxic T-Cells?
- Pathogens that escape the MHC Class II pathway and require cytotoxic T Cells (MHC I pathway)
- Listeria monocytogenes – escape lysosome into cytosol
- Salmonella typhimurium – sequesters antigen from class II pathway
- Epstein Bar virus – suppresses class II expression
What are the two pathways of antigen presentation for MHC Class I and Class II (picture)?

Differentiate the characteristics of innate and adaptive immunity.
- Speed of Response
- Innate is immediate (actually means always present)
- Adaptive is delayed
- Immunological Memory
- Innate response is always the same response
- Adaptive memory response is faster and stronger with greater specificity after each encounter with antigen
Describe how innate immunity is triggered.
Two main triggers
Infection
Tissue damage
What are Pattern Recognition Receptors (PRRs)?
Trigger innate response by binding repetitive structures
What are types of PRR ligands?
- Ligands
- PAMPS (Pathogen-Associated Molecular Patterns): pathogen-induced
- RNA, DNA, polysaccharides, flaggelin
- DAMPS (Damage-Associated Molecular Pattern): autoimmune
- uric acid, cholesterol crystals, DNA, chaperone proteins
- PAMPS (Pathogen-Associated Molecular Patterns): pathogen-induced
What are PRRs located? Why?
- Locations – important because infections/viruses can arise in all three compartments
- Extracellular
- Cytosolic
- Endosomal
What are PRRs classes?
- Toll-Like Receptor (TLRs) at plasma membrane and in endosomes
- C-type lectin receptors (CLRs): recognize carbohydrates at plasma membrane
- Retinoic acid-inducible gene (RIG) Like Receptors (RLRs): recognize viral RNA in cytoplasm
- DNA Sensors: recognize viral DNA in cytoplasm
- NOD-like receptors (NLRs): recognize NLRP-3 domain and peptidoglycans in cytoplasm
How do Toll-Like Receptors Promote Inflammation?
(2) Pathways
- Activation of NF-kB (cytokines) – acute inflammation
- Activation of IFNs (interferons) – resistance to viral infection
- Plasmacytoid dendritic cells produce type I IFNs → expression of interferon stimulated genes → inhibition of viral RNA replication and activation of immune response
- Act in an autocrine and paracrine manner to protect uninfected cells
Describe the NLRP-3 Inflammasome Pathway?
- Phagocytosis by macrophages → recognition of PAMPS/DAMPS → NLRP-3 trimerization with adaptor protein and caspase-1 → cleavage and activation of caspase-1 → caspase-1 cleaves pro-IL-1B → active IL-1B → secretion → fever/acute inflammation
- Can cause auto-inflammatory syndromes, kidney diseases, and gout (by urate crystals)
What are the 2 barriers of the innate immune system?
- First Barrier – epithelium and mucous
- Second Barrier
- Intraepithelial lymphocytes (IELs) patrol epithelial layers
- Have low specificity and thus attack anything foreign
- Produce peptide antibiotics (defensins cathelicidins)
- Intraepithelial lymphocytes (IELs) patrol epithelial layers
What are Neutrophils (multi-lobed)?
- Rapid turnover (short half-life)
- First responders
What are Monocytes?
Explain respitory burst?
- Monocytes (differentiate into Macrophages once in tissues (horse-shoe lobbed))
- Ingest microbes with actin
- Long half-life in healthy tissues
- Macrophage phagocytosis can act with respiratory burst by activation via cytokines (among other pathways to kill)
- Cytokine receptor binds cytokine → NADPH → NADP+ via NADPH oxidase → produces ROS → ROS kills microbes
Chronic Granulomatous Disease
NADPH deficiency leads to macrophage ingesting microorganism without ability to eliminate it
What are denditirc cells?
- Constantly sampling environment with dendrite fingers to look for PAMPs and DAMPs
- Antigen-Presenting Cell (APC) that activates naïve T-cells
What are mast cells?
Abundant cytoplasmic granules that contain vasoactive amines that increase vascular permeability
Produce allergic reactions
What are innate lymphoid cells?
Aren’t T-Cells, don’t have T-Cell Receptors, but have similar actions
What are natural killer cells?
MOA?
What do they release?
- Normal Cell: has activating receptor and inhibitory (self-MHC I receptor) → NK Cell binds but not activated → no cell killing
- Virus-Infected Cell: only has activating receptor and absence of inhibitory receptor (self-MHC I)→ NK Cell activated → cytotoxic granules induce apoptosis
- Produce IFN-gamma (interferon gamma) to induce macrophage killing
- Macrophages release IL-12, IL-15 → recruits NK cells to area
What are Proteins critical for innate immunity?
- Complement – critical for targeting bacteria
- C3b tags microbes for degradation
- Collectins (“sticky”)
- Mannose binding lectin
- Surfactant
- C-reactive protein
- Fibrinogen
- Anti-microbial peptides
- Antibiotic-like properties
- Cytokines
- Cell activation and differentiation
- Chemokines
- Cell recruitment
Describe the sequence of events involved in the inflammatory response.
- Microbe breaches epithelium
- Resident immune cells are triggered by PAMPs to produce cytokines and chemokines
- Cytokines activate endothelium to produce receptors and becomes permeable
- Leukocytes are recruited from the blood into extravascular space
- Chemokines increase integrin affinity with leukocyte
- Binding to selectin initiates extravasation
- Binding integrin ligand has high affinity
- Microbes and dead cells are eliminated in the tissue
- Leukocyte adhesion deficiency
- Deficiency in integrins and selections lead to increased susceptibility to infection

Describe how the innate immune system stimulates adaptive immunity.
Dendritic cells and other Antigen Presenting Cells activate T-Cell differentiation
Describe where and how naïve T cells recognize antigens.
- Once thymocytes leave the thymus, they become naïve T cells
- Naïve T cells live in lymph tissue
- APCs produce peptide + MHC Class I or II protein and migrate from peripheral tissues to lymph nodes
Induction Phase versus Effector Phase
- Induction Phase: naïve T cells bind to APCs via T Cell Receptor to become “activate,” triggering clonal expansion to become effector T cells
- Effector Phase: effector T cells enter into blood, travel to periphery, and activate cell killing and activate macrophages
Describe the events that occur when T cells are activated.

What are the three phases of Signal 1?

What is Signal 2?

Signal 3?

What is the end result of signal transduction?
End result of T cell signal transduction is the activation of transcription receptors including NFAT and NF-kB and activates mTOR protein à increases protein synthesis
NFAT pathway?
Ca++ enters cells → binds calmodulin → which binds calcineurin (a phosphatase) → dephosphorylates NFAT → NFAT acts as a transcription factor → transcribes IL2 → T cell clonal expansion
What is Tacrolimus?
- Tacrolimus – Calcineurin inhibitor
- Prevents rejection of allograft organs
What is Immunodominance?
Immunodominance – most expanded T cells clones recognizes only a few peptides of a given microbe
Effector CD4+ T cell: Helper T cell (2) functions
- Cell mediated immunity
- CD40:CD40L binding → macrophage activation → kill phagocytosed microbes
- Humoral immunity
- CD40:CD40L binding → antibody hypermutation and class switching
Mechanism of Extraversion
Difference between Naive and effector?
- Selectin (T cell) to Selectin ligand (endothelium) binding → weak adhesion between T cell and endothelium
- Integrin (T cell) to Integrin ligand (endothelium) binding → stabilizes adhesion between T cell and endothelium
- LFA-1 (naïve T cell) to ICAM (endothelium)
- VLA-4/ LFA-1 (effector T cell) to VCAM/ ICAM (endothelium)
- Chemokine Receptor to chemokine binding → activation of integrin and chemotaxis towards lymph cortex or site of infection
- CCR7 (naïve T cell) to CCL19/21 (endothelium)
- CXCR3 (effector T cell) to CXCL10 (endothelium)
What are memory T-cells?
3 Types
- After infection, most effector T cells die via apoptosis, some remain (Memory cells)
- Maintenance requires certain cytokines (IL-7 and IL-15) but not any antigen
- Three classes of memory cells
- Central: located in lymphoid organs
- Effector: circulating
- Tissue-resident: mucosal sites
Describe the similarities between a B cell receptor and a T cell receptor.
- Membrane bound
- Recognize linear epitopes
- Have one constant and one variable region on each chain
- TCR looks similar to the Fab region of the BCR
Describe the differences between a B cell receptor and a T cell receptor.
- BCRs have antibodies that can be membrane bound or secreted while TCRs are membrane bound
- BCRs recognize both linear epitopes and conformational epitopes
- BCRs bind free antigens while TCRs bind peptides associated with MHC
- BCRs facilitate signal transduction with Ig-alpha and Ig-beta proteins while TCRs use CD3s and Zeta proteins
- TCRs don’t undergo class switching or affinity maturation (somatic hypermutations)
Antibody structure
- 4 polypeptide chains – 2 heavy chains and 2 light chains
- Heavy chain: 1 variable region and 3 constant regions
- Heavy chain type (i.e. mew, alpha, epsilon) determine functional class
- Light chain: 1 variable region and 1 constant region
- Both light chains must be either lambda or kappa but not both
- Kappa:Lambda:2:1 (distortions may indicate abnormal proliferation of B cell clonal expansion)
- Tail piece at C terminus end of each heavy chain
- Contains transmembrane anchor – B cell receptor
- Lacks transmembrane anchor – secreted antibody
Papain vs. Pepsin
Papain is an enzyme that cleaves the Fab and Fc above the hinge
Pepsin is an enzyme that cleaves the Fab and Fc below the hinge
Describe the importance of the variable and hypervariable regions of an antibody molecule.
- Each variable region of each heavy or light chain has three hypervariability segments and fold to form the antigen binding pocket
- Epitope – the specific region of the antigen that binds to the antibody
Avidity versus Affinity
- Avidity – the strength of all of the antigen binding sites combined
- Monomers (IgG) have two binding sites while pentamers (IgM) have ten binding sites
- Affinity – the strength of one antigen binding site
- Antibodies with low affinity binding sites can have an overall high avidity, depending on the number of these binding sites
Pro vs. Pre B Cell?
Pro-B Cell – germline DNA (progenitor)
Pre-B Cell – recombined DNA of the heavy chain (immature)
What is allelic exclusion?
- Allelic Exclusion
- For both heavy chain and light chain VDJ, only the paternal or maternal allele for each chain protein is recombined at a time
- For example: if the maternal allele for a heavy chain is recombined properly, it will be transcribed. The paternal allele would be excluded from transcription and VDJ recombination
- For both heavy chain and light chain VDJ, only the paternal or maternal allele for each chain protein is recombined at a time
explain VDJ recombination?
- During T and B Cell Receptor somatic recombination…
- VDJ Recombination
- VDJ
- Variable Region (V) – tons of genes for mother and father on different chromosomes
- Diversity Region (D) – tons of genes
- Joining Region (J) – surrounding hinge of AB
- Recombination in germline can make diversified TCRs and antibodies to recognize large number of possible pathogens
- Random process via recombinase enzyme
- VDJ
- VDJ Recombination
Describe the two types of cell-mediated immunity which provide defense against intracellular pathogens.
Name any subtypes.
- T Helper Cells
- TH1 – release cytokines that stimulate macrophages activation to kill ingested microbes via ROS and NO
- TH2 – activates eosinophils and M2 macrophages for tissue repair
- TH17 – release cytokines that stimulate pro-inflammatory pathway, killing microbes
- Cytotoxic T Cells
- Kill infected cells
Describe differences between the presentation of peptides derived from microbes in vesicles and microbes in cytoplasm.
- Phagocytic microbes (MHC Class II/CD4+)
- Proteins are not synthesized by infected cell – it is still exogenous
- Peptides interact with MHC Class II via endosomal/lysosomal binding
- “Cross presentation”
- Cytosolic microbes (MHC Class I/CD8+)
- Proteins are synthesized by host cell – endogenous
- “Direct presentation”
General function associted cytokines fro each Helper t cell subtype.
CD 4+ T cell Type
Defining Cytokines
Target Cells
Defense Against
Th1
IFN-gamma
Macrophages
Intracellular pathogens
Th2
IL-4,5,13
Eosinophils
Parasites
Th17
IL-17,22
Neutrophils
Extracellular pathogens
Th1 synthesis?
What does it release?
Th2 synthesis?
What does it release?
- Synthesis/Differentiation: via IL4 → activate GATA-3
- Releases cytokine: IL4, IL5, IL13
Th17 synthesis?
What does it release?
- Synthesis/Differentiation: via IL6/IL23/TGF-beta → activate ROR-gamma-t
- Release cytokines: IL17,22
Function of IFN-gamma? (2)
- Activates macrophages → microbial killing via ROS
- CD40L needed for activation of macrophage
- CD40L not always present on Thl surface. It is upregulated when Th1 binds to MHC II complex.
- CD40L needed for activation of macrophage
- Activates B cell proliferation / Antibody production
- Upregulation of complement binding and opsonizination (when antigens bind IgG triggering phagocytosis)
Function of IL4?
- Stimulates production of IgE
- IgE → mast cells → histamines → allergic reaction
- Stimulate B cells → production of antibodies (IgE) against helminth worms
Function of IL5?
- Activate eosinophils → bind to IgE (from IL4) → eosinophils release lytic granules → kill worms
Function of IL13?
- In intestines: stimulates mucus secretion and peristalsis → inhibits worm entry and promotes worm expulsion
- Activates alternative macrophages (M2 macrophages) → tissue repair
**** Works with IL4
Function of IL17?
- Act on leukocytes and tissues cells → produces cytokine and chemokines → recruit neutrophils → inflammation
- Good if against bacterial/fungal extracellular infection
- Bad if against endogenous extracellular molecules (i.e. MS, IBD)
Function of IL22?
Act on tissues cells → increased barrier function/wound healing
Explain the TH1/ TH2 balance
- Th2 cytokines inhibit Th1 immune responses
- Th1 cytokine promotes macrophages to kill microbials
- Th2 cytokines inhibit microbial killing
- Normally, Th1 > Th2
Explain how the TH1/ TH2 balance contributes to different outcomes of a Mycobacterium leprae infection.
- Mycobacterium leprae → Leprosy
- Th2 > Th1 → unable to eradicate infection → lepromatous leprosy (destructive lesions)
Th1 > Th2 → activation of T cells and macrophages → tuberculoid leprosy (less destruction form)
Describe mechanisms used by CD8+ T cells to kill infected cells.
- After antigen-binding to effector cytotoxic T cells, prepackaged cytolytic granules are released towards target cell
- Granules are not cell specific, close junction between target cell and T cell reduce effects on endogenous non-harmful molecules
What is the Granule Mech?
- Granule contain Granzyme B and Perforin
- Perforin – perforates transmembrane on target cell
- Granzyme B – cleaves and activates caspases → triggers apoptosis → degrades viral DNA via apoptotic nucleases
What are some other pathways to activate apoptosis by killer T cells?
Fas/Fas Ligand
TRAIL/TRAIL Receptors
Explain the Fas mechanism?
What happens in Cancer?
- Normal: T-Cell expresses FasL and target cell express FasR → FasR binding activates caspases → apoptosis
- T-Cell Downregulation: One T cell expresses FasL and second expresses FasR → FasR binding activates caspases → apoptosis → downregulation of T cell effectors after infection
- Cancer: Cancer cell expresses FasL and T cell expresses FasR → cancer triggers apoptosis of T cells and inhibits immune response
How do pathogens cause T Cell Exhaustion?
Chronic Infections(HIV/HBV)/Tumors repetitively stimulate T cells → T cell exhaustion → inability to respond to viruses
What are the 3 immune mechanisms involved in the response to cancer?
- CD8+ T cell-mediated killing of tumor cells
- Tumor presents MHC I + peptide
- Binding stimulates secretion of IFN-gamma (to block tumor proliferation) and perforin/granzymes (apoptosis)
- CD4+ T-cell mediated control of tumor cells
- Macrophage presents MHC II + peptide
- Binding activates macrophage and releases IFN-gamma
- NK cell-mediated killing of tumor cells
- Loss of inhibitory receptor triggers tumor-killing
- Antibody-mediated response to tumor cells
- AB binds tumor proteins → apoptosis
What are the 3 ways tumors escape the immune system?
Name examples of each way.
- Tumor Directly Escapes
- Antigen loss
- MHC Class I Loss
- Production of Inhibitory Receptors
- Production of Immunosuppressive Cytokines (IL-10 and TGF-B)
- Active Suppression
- Regulatory T cells block effector T cell activation
- Myeloid cells (leukocytes) downregulate T cell response
- Problems with Effector T Cells
- T cell exhaustion
- T cell apoptosis
What is passive therapy underlying immunotherapeutic strategies for cancer?
- Passive therapy – temporary effect (you stop treatment, you stop response)
- Antibody mediated
- Antibodies which lead directly to cell death (trastuzumab)
- Engineered antibodies
- Antibody mediated
What is active therapy underlying immunotherapeutic strategies for cancer?
- Active therapy – induced within the host, durable response
- In situ immunization – activates the immune system against endogenous tumor
What is adoptive therapy underlying immunotherapeutic strategies for cancer?
How does this solve issues with patients who do not have tumor reactive cells?
- Adoptive therapy – transfer of autologous (self) or allogenic (donor) immune cells (can be durable) with anti-tumor activity
How does delivery of cytokines work as an immunotherapy?
Delivery of cytokines (giving IL-2 causes proliferation of T cells, even those without IL-2 receptors)
How does ipilmumab work?
Anti-CTLA-4 (ipilmumab) blocks downregulation of activated T cells
How do Anti-PD-1 drugs work?
- Normally, PD-1 in T cell can bind PD-L1 in tumor and APCs, blocking effector functions
- With treatment, AB is used to block binding, therefore restoring T cell effector function (allows signaling cascade to occur)
- Good for metastatic cancers
How doees Provenge work?
Provenge – antigen from cancer is fused with growth factor to trigger prostate specific T cell response
How do Chimeric Antigen Receptor (CAR) Expressing T Cells work?
What can it treat?
- Building a T cell receptor signaling motif with an antibody specific binding domain
- Combines power of signals with specificity of antibody
- Treats Acute Lymphoblastic Leukemia (ALL)
What are potential adverse events associated with cancer immunotherapy?
- Autoimmunity can result when:
- The tumor antigen is not tumor specific (same expressed protein)
- The antigen is cross-reactive with an off-target unpredicted antigen (different protein but similar epitope)
- Immunotherapy is non-specific, triggering all available immune available immune cells (i.e. anti-CTLA-4)