The association of CSR pathway with the level of IgG class switching in CSR mutated mouse.
Xin Peng Huang
The human immune system is made up of varies of leukocytes to recognized and defend our body from pathogens. In the adaptive immune response, the primary leukocytes are the T-cell and B-cell. The B-cells produce immunoglobulins (Igs) that bind to different receptors form various of pathogens. The B-cell obtain its variation of bind site from VDJ recombination by recombination signal sequences (RSS) and secondary modification by class switching recombination (CSR). The CSR has two pathways to produce different classes of Igs. Both pathway required activation-induced deaminase (AID) to deaminate cytosine and convert it to uracil for error-prone DNA repair. The CSR’s primary pathway is the base excision repair (BER) which employ UNG, APE1, and pS38-AID. The minor pathway called mismatch repair involved MSH2, MSH6, MLH1, PMS2, and EXO1. In this experiment, the goal is to determine the association of genotypes in the unknown CSR mutated mice to the IgG1 and IgM phenotype. The result suggested the mutated mouse is UNG-/- genotype but the phenotype is inconclusive because the IgG level of mutated mouse is higher than the WT mouse with the blockage of the primary CSR pathway.
The first line of the human immune system is the innate immune system formed by leukocytes such as the macrophages and neutrophils. The macrophages can recognize common bacteria molecules and engulf the bacteria for phagocytosis. However, when the innate immune system failed to recognize the pathogens, it triggers the adaptive immune response. (5) The adaptive immune response is formed by varies of leukocytes developed from stem cells in the bone marrow. The leukocytes circulate in the bloodstream and capable of recognized and bind to the molecule that signals for an infection by bacteria or a virus. When the leukocytes detect an infection or a foreign protein, it triggers humoral immune system or cellular immune system. The cellular immune system destroys the infected cells but also recognized and destroy foreign tissues and parasites. The cellular immune system is driven by a class of T lymphocytes (T cells). There is two primary type of T-cells in the cellular immune system, the cytotoxic T- lymphocytes (CTL) which recognized infected cells or parasites using T-cell receptors located in the outer surface of the cytotoxic T-cell. (1) The second primary T-lymphocytes are called the helper T-cell (Th-cell), it produces a cytokine called interleukin (IL). The IL binds to IL receptors to activates signaling pathway to proliferate and differentiate T- cells to effector cells. Th-cells also assisted in proliferation B-cells into effector cells or memory cells in the humoral immune system. (3)
The humoral immune system composed of antibody binding to viruses and bacteria to inactivates them. (4) The antibodies are produced by the B-lymphocytes (B-cells) and primarily required 2 signals for activation. The first signal required the binding of antigen to antigen receptor. The second signal is active by Th-cell membrane bind to CD40 ligand recognizing the CD40 protein on the surface of B-lymphocytes. With the simulation of Th-cell, it induced B-cell for proliferation to become an antibody-secreting effector cell. The antibody secret by matured B-cells are called immunoglobulins (Ig), a Y shape protein consisted of four polypeptide chains. The two-identical light chain located at the tip of the Y which consist of antigen binding site forms the variable domain. (VL) and two identical heavy chain which forms the constant domain (CH) (4) In order for B-cell to generate various of different receptors for infinite pathogens, it employed a specific recombination technique. This random recombination mechanism is called the V(D)J recombination in the light chain because in the light chain it lacks the D segment, and VDJ in the heavy chain, it shuffles variable (V), diversity (D), and joining (J) genes and recombine them. The V(D)J or VDJ recombination is initiated by a double-strand break in the RSS site next to every variable in the VDJ segment with either 12bp or 24 bp apart. However, the V(D)J or the VDJ recombination has its limited due to the random reorganization of the V, D, J gene segment, the limited of V, D, J gene segment by the parental germline, low affinity to Igs, and limited effector functions. (6) Therefore, the light chains in Ig was further modification with a process called called the somatic hypermutation.
The somatic hypermutation is mediated by activation-induced deaminase (AID). The AID deaminates cytosine and converts it to uracil to trigger an error-prone DNA repair in B-cell and manipulate the DNA repair pathway to prevent deoxy-uracil from being repaired and promote mutation of the Ig genes while maintaining the integrity of the genome. In conclusion, the somatic hypermutation promotes affinity maturation. (7) In the Ig CH, the secondary modification initiates in the CH gene called the class switching recombination (CSR). The CSR modified CH genes with the constant region locus sequence. This mechanism is the primary class switch mechanism, it functions as an intrachromosomally deletion and rearranges on the constant regions sequence. During CSR, junctions called Sx (x can be su, sy, sa, se…) are formed on the CH region locus and developed a loop to excised out constant region locus to form a new class of Igs e.g. IgG1. The role of AID is also vital in this process, after transcription of the CH gene sequence, the AlD deaminate cytosine and convert it to uracil to form blunt and staggered double-strand breaks (DSB) on the Sx region and rejoin using S-S recombinant. (6,7) This process creates a deletion in the CH locus and developed a different class of Igs such as IgG1 from IgM which is the default Ig produce from VDJ recombination mechanism in matured B-cell.
There are two pathways in CSR that creates the DSB. The primary pathway used to form a new class of Igs was called the base excision repair (BER). The BER used uracil- DNA glycosylase (UNG) to remove uracil from the AID conversion which created abasic site at the uracil base pair location. After the uracil is removed by UNG1, apurinic-apyrimidinic endonuclease (APE1) excised the abasic residues and “nick” the DNA to form the sequence for new classes of Igs with the help of pS38-AID. The pS38-AID phosphorylation and interaction with APE1 promotes ATM, a DNA damage response kinase for DSBs for repairing the recombining Sx region. Previous research also shown mutation of AlDS38A, a 38th serine residue in AlD switched into Alaine, the result of this amino acid variation prevents the binding to APE1 and inhibits the BER pathway. (6,8) The secondary pathway is call the mismatch repair (MMR) which use MSH2 and MSH6 protein to recruit other protein such as MLH1, PMS2, and EXO1 upon sensing the uracil in the constant region locus sequence created by AID. The proteins recruited by MSH2 and MSH6 created a DSBs and later recombine into a new class of Igs. (6)
In this report, our primary task is to examine mutated mouse with the knock in or knock out gene associated with the CSR pathway. The genotype of the mouse was used to associate with IgG1 and IgM phenotypes. In this experiment, we sacrifice an unknown mutant mouse and extract the blood sample, spleen, and tail sample to exam the genotype and phenotype. The phenotype examine in this experiment are the IgM and IgG1 level in WT mouse and an unknown CSR mutant mouse (EXP). To determine the level of IgM and IgG we employed the ELISA and flow cytometry technique. The ELISA detects the IgM and IgG level in vivo while flow cytometry sense IgM and IgG level in vitro. To detect the genotype of the mutated mice, we employed the PCR and the qPCR technique to amplified and detect specific section of gene coded for CSR pathways. Additionally, protein involved in CSR pathway was determine using the western blot. (6) For expectation, if the BER pathway are block, the IgG level should be lower than the wildtype (WT) because it is the primary pathway for CSR. If genes are knock out in both pathway, we will expect no or no significant IgG level are shown in vivo and vitro. However, if the MMR pathway is block, we will expect minor drop in IgG level in vivo and vitro because it is the minor CSR pathway.
The data from figure A is obtained by cutting both WT mice and EXP mice’s tail and incubated in heat bath with lysis buffer and protease K to digest the tail for DNA. The tail DNA is harvest by adding isopropanol to digested DNA and then the DNA is gathered with the tail fur using shepherd’s hook. The fur was dipped in 70% ethanol and deposit in TE buffer placed in the water bath. The tail DNA of WT and EXP mice was used in PCR contain UNG forward, reverse primer and NEO forward primer to detect the presence of UNG+/+ or UNG-/- genotype. After the PCR is complete, the samples are loaded into 1.5% agarose gel for DNA electrophoresis. The agarose gel in Figure A shown bands of 0.5 kb in WT, 1:10 dilute WT mice DNA, and WT DNA from Chirs’s group act as a control due to multiple failures in PCR. Figure A also demonstrated bands in 0.6kb in both EXP mice DNA and 1:10 diluted EXP mice DNA suggested that one of the genotype of EXP mouse is UNG-/-. (6)
The western blot is employed to determine the genotype of MSH2 by detecting the MSH2 protein in mouse splenocyte cells. The splenocyte cell was harvest from both WT and EXP mice and culture in B-cell medium to multiple the splenocyte cell. To prepare the sample for western blot, the cells are lysis and centrifuge to obtain the protein content within the cell. The concentration of the protein is measured using the standard curve developed in Bradford assay to calculate the concentration of protein in each WT and EXP sample. (Appendix) The equation obtains in the Bradford assay was used to calculate 50ug of protein in each sample and loaded into 8% acrylamide gel for SDS-PAGE. After SDS-PAGE, the 8% acrylamide gel was transfer to a semi-try transfer sandwich.
The sandwich composed of 2 blotting pad and in between the pad is the 8% acrylamide gel sits on top of PVDF membrane to transfer the protein in the gel to PVDF membrane. After the transfer the protein, the PVDF membrane is cut in half at 80 kDa. One half with large molecular weight (MW) was added with anti-MSH2 antibody and anti-tubulin antibody was added to the half with lower MW. The result obtains from filming the PVDF membrane showed MSH2 protein at the 100 kDa and tubulin protein show at 58 kDa. (6)
The qPCR was employed to determine the AID and AlD S38A genotype by amplifying the WT and EXP DNA with a Taq-man probe. When DNA extended in PCR, it breaks down R side of Taq-man probe and releases fluorescence signal. The data in the qPCR graph in Figure C and D was obtain by using WT and EXP tail DNA to perform PCR but with the addition of either WT probe detecting AID+/+ sequence amplification or S38A-AID probe to detect the AlDS38A/S38A sequence during PCR. (6) The result shown in Figure C demonstrated amplification of AID+/+ sequence with high fluorescence signal (EXP DNA maxima (ma) = 1.400 fluorescence(f), WT DNA ma=1375f) in early round of PCR at 23 cycles. The result of figure D demonstrated signals with a low level of fluorescence compared to WT probe. (EXP DNA maxima (ma) = 0.570, WT DNA ma=0.650f) and begin later in the PCR cycle at 25 cycles and 27 cycles respectively.
To determine the IgM and IgG1 in vivo, ELISA was performed using the mouse blood serum. The blood serum used in ELISA was collected using a needle to draw blood from vena cava in WT and EXP mouse. The blood is centrifuged to separate the cell from the serum. The ELISA plate was prepared by adding anti-IgG1 or anti-IgM capture antibody and probe overnight. In each well we loaded dilute the samples and the antibody standards in half the concentration. The IgM-HRP or IgG1-HRP was added to bind to standard, WT, EXP samples to emit fluorescence signal once induced. (6) Similarly to Bradford assay, we used IgG1 and IgM standard to create a standard curve to calculate the IgM and IgG1 concentration as shown in Table A.
To exam the IgM and IgG1 in vitro, flow cytometry was employed using the splenocyte cell. The splenocyte cell was used to form 6 samples and added antibodies to detect IgM and IgG1 level in WT and EXP splenocyte cells. The different antibody is IgG1-APC which bind to IgG1, B220-FITC binds to IgM, DAPI binds to dead cells and a control that has no antibody. The data is processed in FlowJo to determine the IgM and IgG1 level in vitro. (Figure E + Appendix) The result from Figure E shows no significant level of IgG1 level in EXP splenocyte with 0.23% in B220+/IgM1+. However, in WT splenocyte cell there is a significantly higher level of in B220+/IgM1+ compared to EXP cell 3.14%. The result demonstrated no significant level of IgG1 is found in vitro of EXP mouse.
UNG -/-, AID S38A+
WT vs EXP
The data is from Table B was collected from the mutant mouse from the class. The result shows in the T-test show no significant difference between WT and EXP level on IgM and IgG1 level. (IgM: 0.215975> 0.05, IgG1:0.409194>0.05) However, there are significantly different in WT B220+/IgG1. The data showed the WT IgG1 level is the second highest compared to AID S38A/S38A with the value of 1881.117 ug/ml and 383.5629 ug/ml. Data observe from IgM show all sample contain IgM level >500 ug/ml. The B220+/IgG1+ data show the highest percentage of WT with 9.26% and lowest in AlD-/- 0.013%.
With all the data obtained in this experiment, the genotype is suggested to be UNG single knockout. In the PCR data, the WT data suggested the unmutated UNG gene is located at 0.5kb. However, the PCR data from EXP tail DNA shown band at 0.6kb which suggested the UNG gene is replaced by a knockout method using neomycin resistance sequence. Furthermore, the western blot showed positive tubulin protein present in the film act as a control demonstrate the protein are extracted from a cell because of tubulin function as cytoskeleton in eukaryotes cells such as the solenocyte cell. (6) The western blot suggested the presence of the MSH2 protein indicates the MSH2 gene in the MMR is presence because protein required a DNA sequence to be transcript into protein.
The qPCR data suggested AlD+/+ for both WT and EXP using the WT probe that binds to AlD sequence shows high fluorescence signal in the early stage of PCR suggested the AlD sequence are present in WT and EXP DNA. By comparing the S38A probe WT and EXP DNA data the maxima are different by 0.8f and fluorescence display with 2 cycles different with similar acceleration curve. It suggested the EXP DNA possibly have the same genotype as the WT mouse, therefore, no AlDS38A knock in. In conclusion, the genotype suggested by all the collected data is UNG-/-, AlD+/+, MSH2+, and no AlDS38A/S38A knock in. Since the UNG are responsible for the primary CSR pathway, by knocking out this gene, it prevents the BER pathway from generating IgG1. However, the MMR in CSR still can produce IgG1 due to the presence of MSH2. This correlated with figure E, because the MSH2 are founded inside the cell. The concept also reflex flow cytometry experiment, it only probe for antibody in the cell surface or the dead cells. (6)
The presence of IgG1 in ELISA suggested there is another pathway are associated with CSR that turns IgM to IgG1. However, the data obtained from ELISA are not conclusive compared to previous research. Since the UNG is responsible for BER which the main pathway for CSR, we should see the significant decrease in IgG1 level compare to WT in vivo. A possible explanation is that it is caused by human error, such as mislabeling of samples or pipetting error. However, it is also possible the healthy WT mouse doesn’t produce significant Igs due to environment it was grow in. It is also possible the UNG knock out induced syndromes in mouse induced certain diseases that spike the Ig level because, in the sacrificed EXP mouse, we observe the presence of tumor while dissection. To further investigate the reason for the spike of the IgG1 level compared to WT mouse, experiments can be done by testing WT mouse grow in a sterile environment as a control compared to WT mouse grow in non-sterile and mouse with a tumor to identify the possible factors that change the IgG level.
The class data for B220+/IgG1+ in flow cytometry consistency with previous research because the mutation of either BER or MMR pathway should have decreased the IgG1 percentage or no significant IgG1 in CRS compared to WT. However, the T-test suggested no significant difference in IgM and IgG1 level for WT and EXP mouse which contradicted the results from previous research data. (6) To further investigate the unidentical ELISA data, I suggested to repeat the ELISA again to see if the data is consistence with data obtain in this experiment. It is also possible the mean is not reliable due to small sample size and calculation error.
The challenges in this experiment are the multiple failures in PCR, after multiple attempts with different controls, e.g. fresh primers and Taq polymerase. But lastly, the PCR yield positive DNA product using the master mix solution bought by Dr.Vuong. This indicates the failure of PCR is possibly caused by human error. It could be contamination of the reagent or constant pipetting error. For further validation, we can recalibrate the pipets or repeat PCR again with different PCR buffer.
Special thanks to Sabrina.Q for collaborating with the experiment and Chris for the supple of the WT DNA for PCR. Thanks for SA, NA, AC, CP, ESK, DS, KH, OM, AA, DGM, MF, ES, KS for providing experimental data for analysis. A huge thanks to Dr.Vuong for providing PCR master mix for PCR reaction.
1) Lehninger, Albert L., et al. Lehninger principles of biochemistry. W.H. Freeman, 2013. Page 174-178
2) Hewitt, Eric W. “The MHC class I antigen presentation pathway: strategies for viral immune evasion.” Immunology, Blackwell Science Inc, Oct. 2003, www.ncbi.nlm.nih.gov/pmc/articles/PMC1783040/.
3) Alberts, Bruce. “Helper T Cells and Lymphocyte Activation.” Molecular Biology of the Cell. 4th edition., U.S. National Library of Medicine, 1 Jan. 1970, www.ncbi.nlm.nih.gov/books/NBK26827/.
4) Alberts, Bruce. “B Cells and Antibodies.” Molecular Biology of the Cell. 4th edition., U.S. National Library of Medicine, 1 Jan. 1970, www.ncbi.nlm.nih.gov/books/NBK26884/
5) Janeway, Charles A, and Jr. “Principles of innate and adaptive immunity.” Immunobiology: The Immune System in Health and Disease. 5th edition., U.S. National Library of Medicine, 1 Jan. 1970, www.ncbi.nlm.nih.gov/books/NBK27090/.
6) Vuong, Bao, and Chris Li. Vuong, Bao, and Chris Li. MANUAL FOR LABORATORY in BIOTECHNOLOGY BIO48300/A8300 Part II Winter 2018. CCNY, 2018.
7) Maul, Robert W., and Patricia J. Gearhart. “AID AND SOMATIC HYPERMUTATION.” Advances in immunology, U.S. National Library of Medicine, 2010, www.ncbi.nlm.nih.gov/pmc/articles/PMC2954419/.
8) Kenter, Amy L. “Class-Switch recombination: after the dawn of AID.” Current opinion in immunology, U.S. National Library of Medicine, Apr. 2003, www.ncbi.nlm.nih.gov/pmc/articles/PMC4975044/.
BSA (protein level splenocyte cell) .
Splenocyte count from hemocytometer:
ELISA standard curve:
Data from flow cytometry: