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Advanced Immunology

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Title: Advanced Immunology


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Advanced Immunology June 10, 2005 Benjamin
Bonavida, Ph.D. Office CHS A2-060 Tel
x52233 Email bbonavida_at_mednet.ucla.edu
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Figure 1
Smyth, et al., Molecular Immunology, 2005. 42
501-510.
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METHODS OF DETECTION
1. Morphology by microscopy 2. Staining by
acridine orange 3. PI hypoploidy by flow
cytometry 4. TUNEL by cyto flow cytometry 5. DNA
ladder - 3HDNA fragments released DNA Ladder
Double stranded linker DNA between nucleosomes is
cleaved at regularly spaced inter-nucleosomal
sites, giving use to DNA fragments the length of
which represent the length of nucleosomes
(180-200 base pairs). This typical cleavage is
due to activation of an endogenous
endonuclease. Annexin Annexin V FITC is used to
detect apoptotic cells by flow cytometry. Annexin
V exhibits anti-phospholipase activity and binds
to phosphatidyl serine. TUNEL Tdt-mediated dUTP
nick end labeling. Flow cytometry or
immunohistochemical analysis.
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Purification and characterization of cytolytic
and noncytolytic human natural killer cell
subsets Laura Timares Lebow and Benjamin Bonavida
Proc. Natl. Acad. Sci USA Vol. 87 pp.6063-6067,
August 1990
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Target-induced inactivation and cell death by
apoptosis in a subset of human NK cells Anahid
Jewett and Benjamin Bonavida
Jewett and Bonavida, 1996
J Immunol., Feb 1156(3)907-15, 1996
aNK cells were cultured with K562 under
conditions of NK inactivation in the absence or
presence of IL-2 and the cells were processed for
DNA analysis by the PI and TUNEL methods. The
mean value for all three experiments was
calculated. Different donors were used for the PI
and TdT experiments. bThe p value for the
difference between NK control and NK K562 is
0.002 and for the difference between NK K562
and NK K562 IL-2 is 0.04. cThe p value for
the difference between NK control and NK K562 is
0.004 and for the difference between NK K562
and NK K562 IL-2 is 0.208.
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Natural killer cells and their receptors Derek
Middleton, Maring Curran, and Lynne Maxwell
Transplant Immunology 10147-164, 2002
Natural killer (NK) cells have been known for a
long time to be a very important component of the
innate immune system. However, it is only during
the last 10 years that knowledge of their
receptors has emerged. Described in the present
review are those receptor families killer
inhibitory receptor (KIR) (belonging to the
immunoglobulin superfamily), and killer lectin
like receptor (KLR) CD94/NKG2, that both use HLA
as a ligand and have inhibiting and activating
types of receptors, and natural cytotoxic
receptors (NCR) which do not associate with HLA.
Association of the receptor gives rise to either
an inhibiting or activating signal leading to
either failure or success in lysing a target
cell. The KIR receptors are very polymorphic both
in the number of genes expressed in an individual
and the alleles present for a gene. They would
appear to have had a rapid evolution compared to
the CD94/NKG2 receptors. The roles that NK cells
and their receptors have with various facets of
transplantation, disease, pregnancy and control
of virus infection in humans are described.
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Figure 1
Costello, et al, TRENDS in Immunology, 2004.
25(6) 328-333.
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Figure 2
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Inhibitory NK Cell Receptors
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Signaling by Inhibitory NK Cell Receptors
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Recognition of Target Cells by NK Cells
Activating NK Cell Receptors
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General Signaling Mechanism Activating NK Cell
Receptors
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NK Cells in Viral Infections
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Recognition of viral hemagglutinins by NKp44 but
not by NKp30
Tal Arnon, Marina Lev, Gil Katz, Yehudit
Chernobrov, Angel Porgador, and Ofer Mandelboim
Eur. J. Immunol., 312680-2689, 2001
a) 721.221 cells (106 / ml) were incubated
overnight with 100 l / ml of SV-containing
supernatant. Cells (infected or uninfected) were
washed, incubated with various mAb and
stainedeither with FITC-labeled goat anti-mouse
Ig, or with the NKp44-Ig fusion protein followed
by PE-conjugated goat anti-human Fc. MFI
indicates median fluorescence intensity MFI
numbers were rounded to the nearest whole
numbers. Background staining of SV-infected
721.221 and 721.221 cells with the PE-conjugated
anti-human Fc was 4 and 3, respectively. Results
are representative of four independent
experiments.
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NKp44-Ig binding to 293T cells transfected with
Sendai virus HN cDNA. 293T cells were either
transiently transfected with a control PCDNA3
plasmid (293T/MOCK) or with a cDNA encoding for
HN of SV (293T/pca-svh) using the Fugene
transfection reagent (Boehringer Mannheim). At 48
h later cells were stained either with TC-1D6 mAb
or with KIR2DL1, KIR2DS4, CD16 and NKp44
Ig-fusion proteins. MFI indicates median
fluorescence intensity. Controls were the same
cells stained either with FITC-conjugated
anti-mouse antibodies (no mAb), or with
PE-conjugated anti-human Fc antibodies (no
protein). Results are of a representative
experiment out of two performed.
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Natural killer cells are a source of Interferon g
that drives differentiation of CD4 T cell
subsets and induces early resistance to
Leishmania major in mice Tanya M. Scharton and
Phillip Scott
J of Exp Med., 178(2)567-77, 1993
NK cell cytotoxicity in C3H/HeN and BALB/c mice
after L. major infection. NK cell cytotoxic
activity in the popliteal LNs of L.
major-infected C3H/HeN (A) and BALB/c (B) mice
killed. 1 (dark square), 2 (dark triangle), and 3
d (dark circle) after infection, and uninfected
controls (empty circle) was assessed. Specific
cytotoxic activity of popliteal LN single-cell
suspensions was measured against 51Cr-labeled
YAC-1 targets in a standard 4-b chromium release
assay at various effector cell ratios. (C) NK
cell cytotoxicity in the draining LNs measured 1
day after infection. aAsGM1-treated (empty
square), rabbit serum-treated (dark square),
PBS-treated (dark triangle), and uninfected
control (empty circle). The data shown are from
on representative experiment of three performed.
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NK cell cytotoxicity and early parasite burdens
in inbred mice strains. BALB/c, C57BL/6, A/J,
AKR, CBA/J, CBA/CaH, B6C3F1, CBA/N, C3H/HeJ, and
C3H/HeN mice were infected as described in
Materials and Methods. The NK cell cytotoxicity
(A) in pooled draining LN harvested from two mice
of each strain was measured 48 h after infection.
The number of parasites at the site of parasite
challenge (B) was determined by a modified
limiting dilution analysis of individual excised
lesions as described in Materials and Methods.
Results shown are the mean SE of five mice. (C)
Correlation between NK cell cytotoxicity levels
and parasite titers 2 wk later.
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NK CELLS IN CANCER
- Experimental tumor models - Depletion of NK
increases metastases - Administration of
activated NK cells regression - Human
Beginning of immunotherapy - LAK (lymphocyte
activated killer cells) - LAK kill
freshly-derived tumor cells, cell lines, but not
normal cells - 1985 Rosenberh triumphant
results in terminal patients - LAK IL-2
Response in metastatic melanoma and renal
carcinoma - IL-2 mediated toxic effects. Low
doses are used now - No evidence that LAK
prolongs survival
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Fig. 3. SCID-Winn assay of human melanoma cells
and natural killer (NK) and gd T cells s.c.
coinoculation. The figure shows the growth of a
human melanoma cell line injected s.c. in SCID
mice without (?) and with local coinjection of
autologous NK, Vd1 gd T cells, or Vd2 gd T cells
at the concentration of 2 x 106 cells/ mouse (A)
and 5 x 106 cells/ mouse (B). Tumor growth was
measured every 5 days post implant, and tumor
weight was calculated as described in Materials
and Methods. The histograms represent mean of
tumor weight in 20 animal/point in four different
experiments bars, SD. Lozupone et al., 2004.
Cancer Research 64, 378-385.
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Effects of systemic human natural killer (NK) and
gd T cells i.v. inoculation on the growth of the
autologous melanoma in SCID mice. A, effects of
systemic human i.v. inoculation of either human
or NK or gd T cells contemporary to the s.c.
injection of the autologous melanoma in SCID
mice. The figure shows the growth of a human
melanoma cell line injected s.c. in SCID mice
without and with single i.v. injection of the
autologous NK, Vd1 T cells or Vd2 gd T cells,
contemporary to the s.c. injection of the tumor
cells. Tumor growth was measured every 5 days
post implant, and tumor weight was calculated as
described in Materials and Methods. The
histograms represent mean of tumor weight in 24
animals/point in six different experiments bars,
SD. B, effects of systemic NK and gd T cells
single i.v. inoculation regimen on the s.c. take
of the autologous melanoma in SCID mice. The
figure shows the growth of a human melanoma cell
line injected s.c. in SCID mice without and with
single i.v. inoculation of the autologous NK, Vd1
T cells, or Vd2 gd T cells, at the tumor take (10
days after the s.c. implant). Tumor growth was
measured ever 5 days post implant and tumor
weight was calculated as described in Materials
and Methods. The histograms represent mean of
tumor weight in 15 animals/point in five
different experiments bars, SD. Lozupone et
al., 2004. Cancer Research 64, 378-385.
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