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Advanced Immunology

June 10, 2005

Benjamin Bonavida, Ph.D.

Office: CHS A2-060

Tel: x52233

Email: bbonavida@mednet.ucla.edu

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Figure 1

Smyth, et al., Molecular Immunology, 2005. 42: 501-510.

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METHODS OF DETECTIONMETHODS OF DETECTION1. 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 LadderDNA 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.

AnnexinAnnexin : Annexin V FITC is used to detect apoptotic cells by flow cytometry. Annexin V exhibits anti-phospholipase activity and binds to phosphatidyl serine.

TUNELTUNEL : Tdt-mediated dUTP nick end labeling. Flow cytometry or immunohistochemical analysis.

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Purification and characterization of cytolytic and Purification and characterization of cytolytic and noncytolytic human natural killer cell subsetsnoncytolytic human natural killer cell subsetsLaura 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 Target-induced inactivation and cell death by apoptosis in a subset of human NK cellsapoptosis in a subset of human NK cells

Anahid Jewett and Benjamin Bonavida

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.

Jewett and Bonavida, 1996

J Immunol., Feb 1;156(3):907-15, 1996

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Natural killer cells and their receptorsNatural killer cells and their receptorsDerek Middleton, Maring Curran, and Lynne MaxwellTransplant Immunology 10:147-164, 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 NKp30Recognition of viral hemagglutinins by NKp44 but not by NKp30Tal Arnon, Marina Lev, Gil Katz, Yehudit Chernobrov, Angel Porgador, and Ofer MandelboimEur. J. Immunol., 31:2680-2689, 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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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.

Natural killer cells are a source of Natural killer cells are a source of Interferon Interferon that drives differentiation of that drives differentiation of CD4CD4++ T cell subsets and induces early T cell subsets and induces early resistance to resistance to Leishmania major Leishmania major in micein miceTanya M. Scharton and Phillip Scott

J of Exp Med., 178(2):567-77, 1993

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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 CANCERNK 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 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, V T cells, or V 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 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 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, V T cells or V 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 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, V T cells, or V 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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