Download B cell activation and antibody production

Activation and effector
functions of HMI
Hathairat Thananchai, DPhil
Department of Microbiology
Faculty of Medicine
Chiang Mai University
3 August 2015
วัตถุประสงค์
หลังจากชัว่ โมงบรรยายนี ้แล้ วนักศึกษาสามารถ
• อธิบายความแตกต่างของ Thymus-dependent และ Thymusindependent antigen
• อธิบายความแตกต่างของ primary และ secondary antibody
response
• อธิบายขันตอนในการกระตุ
้
้ น naïve B lymphocyte การสร้ างแอนติบอดี
รวมถึงกลไกการทางานของแอนติบอดี
• อธิบายความหมาย การผลิต และประโยชน์ของ monoclonal antibody
Phases of the humoral immune response
Antigen recognition and antigen-induced
B cell activation
• The activation of B cells requires
antigen recognition in lymphoid
tissues.
– Antigens enter secondary lymphoid
organs through the blood or lymph
and bind to the antigen receptor on
specific B cells.
• The activation of antigen-specific B
cells is initiated by the binding of
antigen to membrane Ig molecules.
BCR; B cell antigen receptor complex
Signal transduction by BCR complex
Properties of Thymus-dependent and Thymus-independent
antigens
Distinct B cell subsets mediates different types of
antibody responses
A second signal is required for B-cell activation by either
thymus-dependent or thymus-independent antigens
Role of CR2 and TLRs in B cell activation
T-B collaboration and the
hapten-carrier effect
There are three important
characteristics of antihapten
antibody responses to haptenprotein conjugate
First, such responses require both
hapten-specific B cells and protein
(carrier)-specific T cells
Second, to stimulate a response, the
hapten and carrier portions have to
be physically linked
Third, the interaction is class II
MHC restricted
Routes of antigen entry
•
Microbial antigens commonly
enter through the skin and
gastrointestinal and respiratory
tracts.
•
Antigens are captured by dendritic
cells and transported to regional
lymph nodes.
•
Antigens that enter the blood
stream are captured by antigenpresenting cells in the spleen.
The sequence of events in T cell-dependent antibody
production-1
1). Antigen is taken up by dendritic
cells and presented to helper T cells.
2). Helper T cells are activeated
and induced to express membrane
proteins (CD40L) and cytokines.
3). Activated helper T cells are
instructed to migrate toward the
follicle.
4). B cells process and present
antigen, and migrate toward the T
cell zone.
Migration of B cells and helper T cells and T-B interaction
Mechanisms of helper T cell-mediated B cell activation
AID ; activation-induced deaminase
The sequence of events in T cell-dependent antibody
production-2
5). T and B cells interact at the
T-B interface, and B cells are
activated by CD40L and cytokines.
The sequence of events in T cell-dependent antibody
production-3
6). Small extrafollicular B cell
foci form in the medulla cords
of lymph nodes or between the
periarteriolar lymphoid sheath
and the red pulp of the spleen.
B cells in these foci undergo low
levels of isotype switching and
somatic mutation and short-lived
plasma cells that secrete
antibodies.
The sequence of events in T cell-dependent antibody
production-4
7). Activated B cells migrate back
into the follicle. Germinal centers
form within the follicles.
Affinity maturation, isotype
switching, and the generation of
memory B cells occur primarily in
the germinal center of lymphoid
follicle.
Germinal center reactions in T cell-dependent antibody responses
Affinity
maturation
centroblasts
TFH ; Follicular
helper T cell
An overview of affinity maturation
Ig heavy chain
isotype switching
The sequence of events in T cell-dependent antibody
production-5
8). Long-lived plasma cells are
generated from cells activated
in the germinal center reaction,
and some of these terminally
differentiated plasma cells
migrate to the bone marrow.
Sequence of events in humoral immune responses to T cell-dependent antigens
Primary and secondary humoral immune responses
The principle characteristics of primary and secondary
antibody responses
The humoral immune response is
mediated by antibody molecules
that are secreted by plasma cells
Neutralization of microbes and toxins by antibodies
Neutralization of microbes and toxins by antibodies
The humoral immune response is
mediated by antibody molecules
that are secreted by plasma cells
Monoclonal Antibodies
Pathways of DNA synthesis
SALVAGE PATHWAY
DE NOVO PATHWAY
Phosphoribosyl
pyrophosphate
+
Uridylate
Thymidine
Hypoxantine
TK+ (thymidine
kinase)
Aminopterin
Nucleotides
DNA
HGPRT+
(hypoxanthine
guanine
phosphoribosyl
transferase)
Inject mouse with protein X
Generation of monoclonal antibodies
Myeloma cells
(HGPRT ,Ig-)
unable to grow
in HAT
Mouse spleen cells; some cells
(red) make antibody to X
Mix and
fuse cells
Transfer to
HAT medium
Ascites fluid
Culture single cells in
separate wells
Expand
positive clones
Culture
Test each well for antibody to protein X
Humanized antibody : mAb encoded by a recombinant hybrid
gene and composed of the antigen-binding sites from a murine mAb
suffix = “omab”
suffix = “umab”
suffix = “ximab”
suffix = “zumab”
Applications of monoclonal antibody
• Identification of phenotypic markers unique to
particular cell types
• Immunodiagnosis
• Tumor diagnosis and therapy
• Immunotherapy
• Functional analysis of cell surface and secreted
molecules
Cluster of Differentiation (CD)
• A system used for the classification of
monoclonal antibodies direct against
leukocyte surface antigens.
• HLDA (Human Leukocyte Differentiation
Antigens) Workshops evaluated and
compared many of these antibodies.
–
–
–
–
Cellular distribution
Functional studies
Epitope mapping
Nomenclature : CD
• Recently, more than 350 CD molecules for
human have been identified.
Applications of monoclonal antibody
• Identification of phenotypic markers unique to
particular cell types
• Immunodiagnosis
• Tumor diagnosis and therapy
• Immunotherapy
• Functional analysis of cell surface and secreted
molecules
Immunoassay
Sandwich ELISA
Direct immunofluorescence
Applications of monoclonal antibody
• Identification of phenotypic markers unique to
particular cell types
• Immunodiagnosis
• Tumor diagnosis and therapy
• Immunotherapy
• Functional analysis of cell surface and secreted
molecules
Monoclonal antibodies that recognized tumor-specific
antigens have been used to help eliminate tumors.
Antibodies block
inhibitory signal
to PD-1
T-cell receptor
recognizes
tumour cell
A separate therapy
uses antibodies
that bind PD-1L on
the tumour cell
PD-1L binds
PD-1,
inhibiting
T-cell
response
Tumor cells avoid an immune response by binding PD-1 on the surface of T cells. Antibody
therapies that block PD-1/PD-1L binding can reactivate the T cell’s response to the tumor.
Nature Biotechnology 2012 Aug;30(8);729-730.