Download study of data analysis and interpretation

STUDY OF DATA ANALYSIS AND
INTERPRETATION
1. Recognition of diverse primate MIC molecules by human Vδ1 γδ T cells
specific for MICA and MICB. (A D)C1R cells transfected with the cDNAs for
chimpanzee Patr-MIC1, rhesus monkey Mamu-MIC1, African green monkey
Ceae-MIC2, or owl monkey Aotr-MIC1 expressed the encoded MIC proteins
on the cell surface (filled profiles), as shown by immune fluorescence
stainings and flow cytometry.
Kinetics of HLADM and HLA-DR association in wild-type Pala cells. Cells
were pulse labeled with methionine for 15 min and chased in the presence of
an excess of methionine and cysteine for the indicated periods of time (h).
Class II and DM molecules were immunoprecipitated at each time point from
CHAPS or Triton X-100 solubilized lysates (as indicated above each gel) with
the DR-specific mAb L243 or with a polyclonal rabbit antiserum sp ecific for
the DM heterodimer (αDM) and analyzed by SDSPAGE.
HLA-DM associates with DR in a post-Golgi compartment. Pala cells were
pulsed labeled wit methionine for 15 min and chased for the indicated periods
of time (h). DM-DR complexes were immunoprecipitated at each time point
from CHAPS solubilized lysates with anti-DM (αDM). DM-associated class II
molecules were eluted with 0.5% DOC and the supernatants re-precipitated
with DR β chain-specific mAb HB10A (A) or control mAb W6/32 (B). The
precipitates were either treated with Endo H or mock treated and analyzed by
SDS-PAGE.
HLA-DM associates with αβLIP and αβSLIP complexes. (A) Pala cells were
pulse labeled for 15 min with methionine and chased for 2 h in the presence
of an excess of methionine and cysteine. Class II, DM and I chain molecules
were immune precipitated from CHAPS solubilized lysates with anti-DM
(αDM) or control anti-C23V (αC23V) or from Triton X-100 solubilized lysates
with anti-DM, DR-specific mAb L243, I chain–specific mAb PIN.1.
3. CD4+CD25+ immune regulatory T cells represent a unique lineage of
thymic-derived cells that potently suppress both in vitro and in vivo effectors T
cell function. We analyzed CD4+CD25+ and CD4+CD25− T cells by DNA
microarray, identifying 29 genes differentially expressed in the resting
subpopulations, and 77 that were differentially expressed following activation.
Most of these genes were elevated in the CD4+CD25+ population,
suggesting a previously activated phenotype. Among these were a number of
genes that antagonize signaling, including members of the SOCS family,
which may contribute to their anergic phenotype. Multiple cell surface
receptors also had increased expression in CD4+CD25+ cells,
including GITR, a member of the TNF receptor super family. Importantly,
antibodies to GITR abrogated suppression, demonstrating a functional role
for this receptor in regulating the CD4+CD25+ T cell subset.
4. Immersion of preformed, ordered alkanethiolate SAMs into dilute solutions
of the conjugated molecules results in two-dimensional matrix isolation of
conjugated adsorbates in the host SAM. The post-immersion host SAM
matrix shows retained alkanethiolate ordering with the guest molecules
inserted both singly into boundaries between SAM structural domains and in
bundles at substrate step edges. Inserted molecules of lengths in the 15 Å
range adopt surface orientations similar to those of alkanethiolate molecules
at all compositions, including the pure conjugated SAMs. In contrast, the
configuration of an inserted, long conjugated molecule (40 Å), varies
monotonically with the final SAM composition. When inserted at decreasing
fractions into an n-octanethiolate matrix, the average tilt of the long molecular
axis decreases and approaches alignment with the host SAM. Combined
quantum chemical calculations and IRS data for the short guest−host SAMs
support a picture of a dense local environment of the host SAM around the
guest molecules, despite their insertion at host defects. These results have
important implications for designing electronic devices based on the
addressing of individual, fully conjugated molecules self-assembled at gold
electrodes.
The structure of a series of alcohol-terminated bi-functional long-tail
organosilane films with varying numbers of superimposed monolayer of the
cell, prepared on smooth, hydrophilic silicon substrates by the layer-by-layer
self-assembly approach, has been investigated with the purpose of
elucidating details of the molecular organization and the intra- and interlayer
modes of binding in such films. To this end, experimental results obtained by
synchrotron X-ray scattering, micro-Raman, and Fourier transform infrared
(FTIR) spectroscopic techniques have been combined and compared. A
comprehensive analysis of all data demonstrates that the studied multilayer
films consist of stacks of uncorrelated discrete monolayer, the inner molecular
order of which is preserved with the growing total number of superimposed
layers. Similar to self-assembled films of long-tail silages with terminal
−COOH groups,1 the intermolecular binding in the present films is
characterized by partial intra- and interlayer covalent bond formation. The
molecular hydrocarbon tails are perpendicularly oriented on the layer planes,
forming a densely packed rotator phase like hexagonal lattice with a
molecular surface area of 20 Å2 and a lateral correlation length of the order of
16 molecular diameters.
5. The intracytoplasmic forms of class II (or Ia) major histocompatibility
complex heterodimers are associated with a third glycoprotein, termed the
invariant chain (Ii). This specific interaction has led to the view that Ii plays a
necessary role in the assembly, intracellular transport, and/or membrane
insertion of Ia molecules. To test this hypothesis directly, we have transfected
complementary DNA clones that encode murine class II alpha and beta
chains into cells that do not express any endogenous Ii messenger RNA
(mRNA).
cDNA encoding the human interferon-γ receptor was isolated from a λgt11
expression library using a polyclonal antireceptor antiserum. The gene for this
receptor was identified in a cosmid library and transfected into mouse cells.
The human interferon-γ receptor expressed in mouse cells displayed the
same binding properties as in human cells. However, transfected cells were
not sensitive to human IFN-γ, suggesting the need for species-specific
cofactors in receptor function. As inferred from the cDNA sequence, the
human interferon-γ receptor shows no similarities to known proteins and
represents a novel transmembrane receptor. It is most likely the product of a
single mRNA and a gene located on chromosome 6.
6. gene targeting techniques to produce mice lacking the invariant chain
associated with major histocompatibility complex (MHC) class II molecules.
Cells from these mice show a dramatic reduction in surface class II, resulting
from both defective association of class II alpha and beta chains and
markedly decreased post-Golgi transport. The few class II alpha/beta
heterodimers reaching the cell surface behave as if empty or occupied by an
easily displaced peptide, and display a distinct structure. Mutant spleen cells
are defective in their ability to present intact protein antigens, but stimulate
enhanced responses in the presence of peptides. These mutant mice have
greatly reduced numbers of thymic and peripheral CD4+ T cells. Overall, this
striking phenotype establishes that the invariant chain plays a critical role in
regulating MHC class II expression and function in the intact animals.
8. We have analyzed the ability of major histocompatibility (MHC) class II
molecules to capture proteins in the biosynthetic pathway and whether this
may be associated with MHC class II-dependent antigen processing. When
co-expressed with HLA-DR 4 molecules in HeLa cells, influenza
hemagglutinin was inhibited from folding and trimerization in the biosynthetic
pathway, targeted to endosomal compartments, and rapidly degraded. Due to
the interaction with MHC class II molecules, therefore, unfolded forms of
hemagglutinin were bypassing the quality control mechanism of the secretary
pathway. More important, however, the transport, endocytosis, and rapid
degradation of unfolded hemagglutinin in the presence of MHC class II
molecules suggest that proteins captured in the endoplasmic reticulum by
class II molecules may become substrates for antigen processing and
presentation to CD4-positive T cells. In insect cells we show that this
phenomenon is not restricted to a few proteins such as hemagglutinin. A
highly heterogeneous mixture of proteins from the endoplasmic reticulum
including co-expressed hemagglutinin can form stable complexes with soluble
HLA-DR α and β chains that were transported into the supernatant. This
mechanism may gain biological significance in abnormal situations
associated with accumulation of unfolded or malfolded proteins in the
endoplasmic reticulum, for example during viral infections.
9. Most protein antigens cannot elicit a T-cell response unless they are
processed to peptides, which are then presented to T lymphocytes by surface
MHC class II molecules. Recent evidence supports an essential role of the
invariant chain associated with class II MHC polypeptides in antigen
processing. Mounting evidence indicates that -GalCer responsive T cells are
heterogeneous in their reactivities to cellular antigens, suggesting that an
individual semi-invariant TCR may be capable of recognizing more than one
ligand. Recent crystal structures of CD1b molecules with three different
bound lipids indicate that the antigenic features of lipids may be localized
over a smaller area than those of peptides, and that the positioning of the
polar head group can vary substantially. A model that explains how CD1drestricted T cells could possess both conserved and heterogeneous antigen
specificities, is that different lipid antigens may interact with distinct areas of a
TCR due to differences in the positioning of the polar head group. Hence,
canonical CD1d-restricted TCR could recognize conserved antigens via the
invariant TCR chain, and have diverse antigen specificities that are conferred
by their individual TCR chains.