Immunology Lab Report 5－ianroo

OBSERVATION OF B CELL ACTIVATION UPON CONTACT WITH CD40L, IL-4, OR BOTH
BY SEPARATION OF CD23 USING GEL ELECTROPHORESIS AND WESTERN BLOT
ANALYSIS

Hsinwei Hu
2007. 04. 28

ABSTRACT
In this lab we examined CD23 expression on activated B cells in contact with either CD40L,
IL-4, or both, by using polyacrylamide gel electrophoresis to separate CD23 proteins, and
Western blot technique to visualize the CD23 band. We observed more intense CD23 band
of B cells in contact with both CD40L and IL-4 than only one of which was present. B cells
without CD40L and IL-4 were found to have a low level of CD23 expression.

INTRODUCTION
The full activation of B cells requires at least 2 of these 3 signals – binding of an antigen to B cell surface Ig, binding of CD40 on B cells with CD40L on activated CD4 TH cells, or the stimulation of cytokines like IL-4. A most common mechanism is: B cells first encounter foreign antigen in peripheral lymphoid tissues, which stimulates resting B cells to enter cell cycle. Then, the internalized protein antigen gets processed and presented on MHC class II on B cells to CD4 TH cells, and thus activates the resting T cells. Activated CD4 TH cells then express CD40L on cell surface, as well as produce the cytokine IL-4. The binding of CD40 on B cells to CD40L on T cells, and the IL-4 receptor on B cells with IL-4, serves as the 2nd and 3rd signal for B cell activation. Upon full activation, B cells express CD23 (“low affinity FceR11 receptor”) on cell surface, which is a marker for activated B cells [1].
In this lab, we will examine the degree of CD23 expression on a human B cell line (Ramos 2G6) under different stimulatory conditions. In each case B cells will contact with nothing, or either activated T cells (expression CD40L) or IL-4, or both. The techniques we will use are Bradford Assay, the polyacrylamide gel electrophoresis (PAGE), followed by Western Blot analysis. The purpose of each technique is as follows:

Bradford Assay – Using several dilutions with known protein concentration, and record their absorbance with a spectrophotometer. Then we can use the plot of absorbance-concentration from the known dilutions to determine the protein concentration of our samples.

PAGE – This is to separate the proteins according to their size. SDS (a detergent) will be added to each protein sample to coat them with a negative charge, so the proteins will migrate to the positive charged electrode end.

Western Blot Analysis – This is to specifically visualize the CD23 band using secondary anti-CD23 rabbit antibodies, and anti-rabbit goat antibodies. The goat antibodies are associated with HRP, an enzyme that emits light while converting the substrate. We will capture this light as chemifluorescent detection.

MATERIALS & METHODS

Isolation of Cytosolic Extract – We set up 4 different cell cultures co-cultured with Ramos B cell. RIPA buffer was added to each cell group to maintain a preferred environment for cells.:

Group Label	Co-cultured Cells	Stimulatory Factor(s)
A	293 cells (kidney epithelial cells)	None. This is the control.
B	293 cells + IL-4	IL-4 cytokine
C	T-BAMS (stimulator T cells)	CD40L expressed on T-BAMS
D	T-BAMS + IL-4	Both CD40L and IL-4

Bradford Assay – We used BSA (a protein) and set up several standard dilutions with known amount of proteins. The concentration of original BSA was 1.45μg/μL.

Then we diluted each sample extract (group A~D) into 2 concentrations:
0.5 μL extract + 799.5μL H2O  2000X dilution
1.0 μL BSA + 799μL H2O  1000X dilution
In each dilution above, 200μL Bradford reagent (a light-blue dye) was added to achieve a total volume of 1mL. Then we used a spectrophotometer to record the absorbance of all the dilutions, and plotted a graph of absorbance-concentration from the standard dilutions. The concentration of the extracts was calculated as follows:
[extract] = extract absorbance × slope of plot × 1mL/1000μL × dilution factor
After each of the concentration was calculated, we got the volume needed to contain 10μg of protein. This was the amount that loaded in PAGE.

PAGE & Western Blot– We loaded 10μg of protein from each extract into the separating gel with 1X tris/glycine loading buffer. The buffer contained SDS which would coat the proteins with negative charges to facilitate migration. It also contained glycine to help proteins “sink” to the bottom of the wells. Before loading, all samples were heated in order to denature the proteins. A “rainbow” ladder with known protein sizes, each represented in a different color, was loaded into the gel to help us identify the sample protein size. The gel was run for about 1 hour. After running, the proteins on the gel were transferred onto a PVDF membrane with Western blot papers.

Chemifluorescent Detection – The membrane with proteins (invisible) was visualized during this process. The membrane prepared above was incubated with milk (to maintain cell environment) and anti-CD23 rabbit antibody (the 1。antibody) and then washed with excess TBST (Tris buffered saline with Tween). The wash step was to eliminate non-specific rabbit-CD23 binding. Later, an anti-rabbit goat antibody (2。antibody )was added, incubated, and washed as well. Then the detection reagent was added to the membrane, folded in a saran wrap. The folded membrane was exposed onto an autoradiography film in the darkroom to visualize the protein bands.

RESULT
The concentration of samples (group A~D) were calculated according to the formula mentioned above. The following was the plot of known BSA concentration with according absorbance during the Bradford Assay. The slope was calculated as 15μg/mL.

A sample calculation for concentration of group A is as following:

The slope of absorbance-concentration plot was 15μg/mL.
The absorbance of 2000X / 1000X dilution were 0.549 / 0.657.
[protein]2000X = 0.549 ×15 μg/mL ×1mL/1000μL × 2000 = 16.47 μg/μL
[protein]1000X = 0.657 ×15 μg/mL ×1mL/1000μL × 1000 = 9.855 μg/μL
[protein]avg = (16.47 + 9.855) / 2 = 13.16 μg/μL
Volume of 10μg protein required = 10μg / 13.16μg/μL = 0.76μL

The total volume of materials loaded into the gel was 10μL, which included 2μL loading buffer, the protein with volume calculated above, and H2O (8-protein volume).

Fig.2 was the result film from the exposure of the membrane prepared in the chemifluorescent detection step. We had labeled the 50kDa and 35kDa bands in the ladder lane (leftmost). The CD23 protein was known to be about 45kDa. The lanes from left to right were group A, B, C, D accordingly. In each lane we observed 2 bands looked like a doublet between 50kDa and 35kDa, which might be the CD23 band we’re looking for. The bands in land D (rightmost) were the most intense ones. Overall, the intensity of bands in the lanes was D > C > B ≒ A.

DISCUSSION
In the Bradford Assay, the plot we got in fig. 1 was approximately a straight line, which was expected since the BSA protein concentration was known and manually controlled. In table. 1, we found that protein concentration in the sample extracts varied between group A, B and C, D. C and D apparently had less protein. However, the protein concentration detected here wasn’t only the CD23 protein concentration, but all the proteins presented around. In the chemifluorescent detection part we shall see that the intensity of CD23 band didn’t parallel with the concentration calculated here in table. 1.
In fig. 2, we were looking for “one” band at 45kDa, which was the known CD23 protein size. We observed 2 bands of equal intensity in between 50kDa and 35kDa, which was not as expected. However, this might due to the presence of 2 isoforms of CD23. Human CD23 was found to have 2 isoforms, CD23a and CD23b. CD23a was induced by antigen activations, whereas CD23b expression was specifically induced by IL-4 [2] [3]. They only differed in 6~7 amino acids in the N terminus. CD23a was largely restricted in B cells, and CD23b was presented and induced in B cells and other haematopoietic cells too. Hence, we might infer that the doublet we found can both be identified as CD23 bands.
Regarding to the band intensity, in fig. 2 we observed the intensity order of D(CD40L+IL-4) > C(CD40L only) > B(IL-4 only) ≒ A(no stimulatory substances). This was quite consistent with our prediction. The expected result was that, B cells with both CD40L and IL-4 stimuli would express more CD23 on cell surface than any other conditions. B cells with only CD40L or IL-4 would have less expression of CD23 comparing to the presence of both factors (however, which condition alone would induce more expression than the other was not differentiated). And finally, B cells without CD40L and IL-4 shouldn’t express CD23 since no stimulatory factors were present. However we still observe a faint doublet around 45kDa in fig. 2. Since the cell culture we used was from a cell line, there might be some basal level of CD23 expression on B cells by some stimulation in the culture media.

REFERENCES
[1] L. Covey, D. Denhardt, B. Firestein, M. Kiledjian, and G. Xiao. Immunology Laboratory Manual. Rutgers University, spring 2007.
[2] Imgenex Co. Rabbit Monoclonal Antibody to CD23, B Cell (Clone SP23) http://www.imgenex.com/abtds_IMG-80345_CD23,+B+Cell.html>
[3] Sophia N. et al. Endocytosis and recycling of the complex between CD23 and HLA-DR in human B cells. Immunology, 2001.