Validating Differences Between Fresh And Frozen Cells

Published Date: 02 Nov 2017

The project involved 9 JSLE patients, 9 JIA inflammatory control patients and 9 patients for the healthy control group. Of the JSLE group the mean age was 15.5 (range 9.6-17.7), the male: female ratio 2:7(22.2% male) and the group was made up with 7 British, 1 Pakistani and 1 Indian. Of the JIA patients studied the mean age was 12.5 (range 6.4-15.1), the male: female ratio 2:7(22.2% male) and all were of white British ethnicity. The healthy control group had a mean age of 12.5 (range 8.7-15.7), the male: female ration 6:3(66.7% male) and all were of white British ethnicity.

Of the JIA group patients studied 1 (11%) had Systemic Arthritis, 4 (44.5%) had Polyarticular Arthritis and the remaining 4 (44.5%) had Oligoarticular Arthritis. Disease activity measured by the number of joints with limited motion was a mean of 3.5 (range 1-7) and mean number of swollen joints was 3.5 (range 1-7). Of the 9 JIA patients 8 were on Steroids for medical treatment.

Data on the medication used on the JSLE group is presented in Table 1. Patients on Retuximab were excluded from the study as this induced a decrease in B lymphocyte count.

Table 1: JSLE Medications

Current medications,

No. JSLE patients* (%)

Hydroxychloroquine

66.7

Azathioprine

11

Mycophenolate

66.7

Prednisolone

44

*Juvenile Systemic Lupus Erythematosus (JSLE) patients may be on >1 of the medications

3.2 Validation and Optimisation

3.2.1 Validating differences between fresh and frozen/thawed cells.

The expression of the three receptors; BCMA (figure 4), BAFF-R (figure 5) and TACI (figure 6) were tested of on fresh and frozen PBMC samples. Experiments were used to validate whether there was a significant difference in protein expression and cell viability detected when cells had been thawed from frozen. There was no significant difference in expression of BCMA(figure6) and BAFF-R (figure 7) between fresh and frozen samples.

TACI expression levels however seemed to alter following cryopreservation (figure 8).

Figure 1: BCMA receptor expression in fresh and frozen/thawed samples

Fresh

Frozen/Thawed

Flow cytometry histograms showing the expression of BCMA receptor in peripheral blood mononuclear cells (PBMCs) isolated from fresh heparinised blood and from PBMCs which had been thawed following freezing. Isotype controls were run and shown overlaid. After washing, cells were stained with an antibody against human BCMA. Isotype controls and unstained controls were run and shown overlaid The BCMA antibody is conjugated to a FITC fluorochrome, detected in the FL1 channel. Graph shows percentage of positive expression for FITC and thus the percentage of positive expression the BCMA receptor.

Figure 2: BAFF-R expression in fresh and frozen/thawed samples

Fresh

Frozen

Flow cytometry histograms showing the expression of BAFF receptor (BAFF-R) in peripheral blood mononuclear cells (PBMCs) isolated from fresh heparinised blood and from PBMCs thawed following freezing. After washing, cells were stained with an BAFF-R mAb. Isotype controls and unstained controls were run and shown overlaid. The fluorochrome conjugated to BAFF antibody was FITC, detected in the FL1 channel. Graph shows percentage of positive expression for FITC, thus showing the percentage of positive expression for BAFF-R.

Figure 3: TACI receptor expression in fresh and frozen/thawed samples

Fresh

Frozen

Flow cytometry histograms showing the expression of TACI receptor in peripheral blood mononuclear cells (PBMCs) isolated from fresh heparinised blood and from PBMCs thawed following freezing. After washing, cells were stained a TACI mAb. Isotype controls and unstained controls were run and shown overlaid. Graph shows percentage of positive expression for PE, the fluorochrome conjugated to the TACI antibody, detected in the FL2 channel, thus showing the percentage of positive expression for TACI.

3.2.2 Different batches of TACI antibody.

Because some variance in expression was found TACI in fresh vs frozen samples, tests were carried out to assess if any differences in TACI receptor expression were caused from using two separate batches of the TACI antibody. Both anti-TACI mAb from separate batches were tested on the same PBMC sample. No variance in TACI receptor expression was detected by the use of different batches of antibody.

Figure 4: TACI receptor expression using two separate batches of antibody

Flow cytometry histograms showing the expression of TACI receptor in peripheral blood mononuclear cells (PBMCs). After washing, cells were stained with TACI mAb. Cells were stained with two separate batches of TACI antibody and an Isotype control and were analyzed by flow cytometry. Graph shows results overlaid an unstained population, representing the percentage of positive expression for PE, the fluorochrome conjugated to the TACI mAb in both the old and new batch of mAb.

3.2.3 Fluorescence Compensation

Compensation is setup on the flow cytometer to remove the fluorescence signal from a given fluorochrome from interfering with other neighbouring channels. Compensation for the CD19 antibody and all three BAFF receptor antibodies was performed using an adult control sample.

Figure 5 Application of fluorescence compensation

Compensation is applied to the flow cytometer to subtract a portion of one detector's signal from another, correcting any fluorescence ‘spillover’. Cells were immunostained with the CD19 antibody (B) and the three BAFF receptors (C, D, E) and compensation was applied to their corresponding fluorchoromes and the channels they are detected by. The results are shown as dot plots and samples in figure 8 are derived from adult control PBMCs.

3.3 Measuring expression of the BAFF receptors in B cells and non B cells.

The results outlined here refer to experiments conducted to measure the variance in expression levels of the three BAFF receptors, in the JSLE group, JIA group and healthy control group. Receptor expression was measured in B cells, positive for the CD19 marker and in the remainder CD19 negative cells Isotype expression for each receptor was also tested and receptor expression was measured using flow cytometery, as exemplified in figure 11. The Isolated PBMCs were thawed from frozen for all patient samples apart from 2 patients in the JSLE group (L50M and L45Q) which were fresh PBMC samples.

Figure 6: Dot plot resulting from a flow cytometry analysis of PBMCs, showing unstained sample, Isotype control expression and BAFF-R expression.

A. Unstained sample B. Isotype Control C. BAFF-R and CD19 stained

Representative dot plot from analysis of PBMCs by flow cytometry, showing how percentage of receptor expression was measured. PBMCs were thawed, washed and stained with fluorochrome conjugated monoclonal antibodies (mAb) A. PBMC population not stained with a mAb, percentage of unstained cells shown in the bottom left (Q4) quadrant. B. PBMCs stained with BAFF-R Isotype control mAb, percentage of cells with positive expression for Isotype control is shown on the X-axis shown in the bottom right (Q3) quadrant and shows no / little unspecific binding C. PBMCs were dual stained with a BAFF-R mAb (FITC labelled) and CD19 mAb (PECy5 labelled). BAFF-R protein expression/ (FITC detection) is depicted on the X-axis, while positive expression for CD19(PECy5 detection) is shown on the Y-axis. Thus, the percentage of cells which are B cells/ CD19+ and express BAFF-R is shown in the top left (Q2) quadrant and percentage of non-CD19+/ CD19- cells which express BAFF-R is shown in the Q3 quadrant. This sample of PBMCs isolated derives from a JIA (J60a) patient.

3.3.1 BAFF-R protein expression in PBMCs

BAFF-R protein expression was determined by FITC labelled BAFF-R antibody detection via flow cytometry (Figure9). Any positive Isotype control expression was subtracted from positive fluorescence values for BAFF-R. It was found that BAFF-R expression was statistically higher in CD19- cells of JSLE than controls (P=0.047). BAFF-R expression was also notably higher in CD19- cells in JSLE patient’s (P=0.0502) than JIA. BAFF-R expression was higher in JSLE CD19+ cells to that on control and JIA patients but this was not statistically significant.

Figure 7: Variation in BAFF-R expression in CD19+ B cells and CD19-PBMCs in JSLE and JIA patients and healthy controls.

PBMCs isolated from Juvenile onset Systemic Lupus Erythematosus (JSLE) (n = 9) and Juvenile Idiopathic Arthritis (JIA) (n = 9) patients and non-inflammatory healthy controls (n = 9). Cells were dual stained with BAFF-R mAb and a CD19 mAb, a marker for B cells, and expression was measured using flow cytometery. BAFF-R expression was statistically higher in CD19- cells of JSLE than controls (P=0.047) but not statistically significant in CD19- cells. BAFF-R expression was notably higher in CD19- cells is JSLE patient’s (P = 0.0502) than JIA however this difference did not reach statistical significance. BAFF-R expression was higher in in CD19+ cells of JSLE than JIA patients but not statistically significant. P-values were determined using Mann-Whitney U test.Values are the mean ± SEM.

3.3.2 BCMA protein expression in PBMCS.

BCMA protein expression was determined by FITC labelled BCMA antibody detection using flow cytometry (Figure10). Isotype control expression was subtracted from positive fluorescence values for BCMA. BCMA expression was statistically higher in CD19- cells of JSLE than controls (P=0.0315) and there was a similar trend in CD19+ cells(P=0.06), although not of statistical significance. BCMA expression on CD19+ cells was higher in JSLE patients than JIA (P=0.13) and CD19- cells (P=0.25) but not statistically significant. When data from two samples with abnormally high (>40% BCMA expression) was removed (figure 14) BCMA expression on CD19- cells was also statistically higher in JSLE than JIA patients (P=0.04)

Figure 8: BCMA expression levels in CD19+ B cells and CD19-PBMCs in JSLE and JIA patients and healthy controls.

PBMCs isolated from Juvenile onset Systemic Lupus Erythematosus (JSLE) (n = 9) and Juvenile Idiopathic Arthritis (JIA)(n = 9)patients and non-inflammatory healthy controls (n = 9) were thawed from frozen. Cells were dual stained with BCMA mAb and CD19 mAb, a marker for B cells, and expression was measured using flow cytometery. BCMA expression was statistically higher in CD19- cells of JSLE than controls (P=0.0315) and notably higher in CD19+ cells, however this difference did not reach statistical significance(P=0.06). BCMA expression was higher in JSLE patients than JIA in CD19+ cells (P=0.13) and CD19- cells (P=0.25) but not statistically significant. P-values were determined using Mann-Whitney U

test.Values are the mean ± SEM.

Looking at the raw data (appendix) some patients samples produced abnormally high BCMA expression levels in the CD19- population of cells, with expression values >30%. These values deviated from all other standard BCMA expression values in CD19- populations of cells. Thus the data for patients J70A (43.1%), L3L (42%) were excluded and results shown (figure 14).

Figure 9: BCMA expression levels in CD19+ B cells and CD19-PBMCs in JSLE and JIA patients and healthy controls, excluding 3 patient samples.

PBMCs isolated from Juvenile onset Systemic Lupus Erythematosus (JSLE) (n = 8) and Juvenile Idiopathic Arthritis (JIA)(n = 8)patients and non-inflammatory healthy controls (n = 9) were thawed from frozen. Data from 1 JSLE, 1 JIA and 1 healthy control was excluded as expression values were regarded as atypical. Cells were dual stained with BCMA mAb and CD19 mAb, a marker for B cells, and expression was measured using flow cytometry. BCMA expression on CD19- cells was higher in JSLE than controls (P=0.0592) asnd was statistically higher than JIA patients (P=0.04). BCMA expression was higher in of JSLE CD19+ cells than control and JIA but not of statistical significance.

3.3.3 TACI protein expressionn in PBMCS

TACI protein expression was determined by PE labelled TACI antibody detection using flow cytometry (Figure10). Isotype control expression was subtracted from positive fluorescence values for TACI. TACI expression was higher in JSLE samples than controls and JIA patients in both CD19- and CD19+ populations of cells but not statistically significant.

Figure 10 Variation in TACI expression in CD19+ B cells and CD19-PBMCs in JSLE and JIA patients and healthy controls.

PBMCs isolated from Juvenile onset Systemic Lupus Erythematosus (JSLE) (n = 9) and Juvenile Idiopathic Arthritis (JIA)(n = 9)patients and non-inflammatory healthy controls (n = 9) were thawed from frozen. Cells were dual stained with TACI mAb and CD19 mAb, a marker for B cells, and expression was measured using flow cytometery. TACI expression was higher in JSLE samples than controls in both CD19-(P=0.22) and CD19+(P=0.33) populations of cells but not statistically significant. TACI expression was higher in JSLE samples than JIA samples in both CD19-(P=0.16) and CD19+(P=0.3) populations of cells but not statistically significant. P-values were determined using Mann-Whitney U test.Values are the mean ± SEM.

3.3.4 BAFF Receptor expression in JSLE patients

The variation in expression levels of BCMA, BAFF-R and TACI are shown for JSLE patients. There was statistically higher BAFF-R expression than TACI expression on B cells (P= 0.0142) as well as CD19- cells(P=0.0019). CD19+ cells also appeared to expressed more BAFF-R. than BCMA. Previously, the values for BCMA expression which were abnormally high were removed and the revised data was also presented. Thus the BCMA data for patient J70A has been excluded and results also shown (Figure16.B).Again, CD19- cells showed highest BCMA expression that was significantly higher than TACI expression (P=0.0037).

Figure 11: Variation in expression levels of BCMA, BAFFR and TACI receptors in CD19+ B cells and CD19-PBMCs in JSLE patients.

A

B

Expression profiles of the three BAFF receptors vary in JSLE patients. A. PBMCs were isolated from Juvenile onset Systemic Lupus Erythematosus(JSLE) patients (n = 9). Equal numbers of cells were aliquoted per condition and samples were immunostained separately with BCMA, BAFF-R and TACI mAb and all were dual stained with a CD19 mAb. Expression was measured using flow cytometery. There was statistically higher BAFF-R expression than TACI expression in CD19+ cells (P= 0.0142) and CD19- cells (P=0.0019).BAFF-R expression on CD19+ cells was also higher than BCMA expression but not statistically different. BCMA expression was higher than TACI expression with statistical significance in CD19- cells(P=0.0019). B. BCMA (n=8) data from one patient was removed, BCMA expression in CD19- cells was still statistically higher than TACI expression (P=0.0037) P-values were determined using Mann-Whitney U test.Values are the mean ± SEM

3.4 Measuring expression of the BAFF receptors on T cells

Results showed that CD19- negative populations of cells also expressed BAFF receptors. Thus a further investigation was set up to look at which of the CD19- cells may have receptor expression.

Expression of BAFF-R, BCMA and TACI was measured on T cells, which express the CD3 marker. Expression was compared in JSLE patients (n=2), JIA patients (n=2) and healthy control patients (n=2). Isolated PBMCs which had undergone cryopreservation were thawed, washed and stained with fluorochrome conjugated monoclonal antibodies (mAb) for BCMA, BAFF-R and TACI and dual stained with a CD3 mAb. Receptor expression was measured using flow cytometery. All three receptor were detected on T cells. BAFF-R was detected in JIA, JSLE patients and low levels in control patients. BCMA expression was highest in JIA samples with controls showing little expression. TACI expression was higher in control samples than JIA and JSLE samples. No statistically significant trends were found.

Figure 12: BAFF receptor expression on CD3+ T cells in JSLE, JIA and healthy control patients.

B

A

C

PBMCs isolated from Juvenile onset Systemic Lupus Erythematosus (JSLE) (n = 2) and Juvenile Idiopathic Arthritis (JIA) (n = 2) patients and non-inflammatory healthy controls (n = 2) were thawed from frozen. Equal numbers of cells were aliquoted per condition and samples were stained with BCMA, BAFF-R and TACI mAb and all were dual stained with a CD3 mAb. Expression was measured using flow cytometery. A. BAFF-R expression on CD3+ cells was higher in the JIA and JSLE patients than the healthy controls. B. BCMA expression on CD3+ cells was highest on JIA samples and low on healthy controls. C.TACI expression was higher control samples than in the JIA and JSLE samples. Values are the mean ± SEM

3.5 Measuring expression of the BAFF receptors on Neutrophils

As some receptor expression was found on T cells, BAFF-R, BCMA and TACI, neutrophils of an adult healthy control were also looked at for receptor expression. Seeing as these cells produce BAFF, it is possible that they may also express receptors for BAFF. Some receptor expression was found (figure 18), although overall expression levels were low, there was some notable expression of the TACI receptor. Thus, receptor expression on neutrophils of JSLE patients was also looked at (figure 19). Low expression was detected for all three BAFF receptors and a slightly higher expression of the TACI receptor was detected.

Figure 13: expression of the BAFF receptord on neutrophil cells of an adult healthy control.

Neutrophils were isolated from an adult healthy control (n=1). Equal numbers of cells were aliquoted per condition and samples were stained with a BCMA, BAFF-R or TACI mAb and expression was measured using flow cytometery. Moderat levels of TACI receptor expression was detected

Figure 14: Expression of the BAFF receptors on neutrophil cells of JSLE patients.

Neutrophils were isolated from Juvenile onset Systemic Lupus Erythematosus patients (n=2). Equal numbers of cells were aliquoted per condition and samples were stained with a BCMA, BAFF-R or TACI mAb and expression was measured using flow cytometery. Low levels of expression were detected for all three of BAFF receptors, but slightly higher expression of the TACI receptor was detected.

4. DISCUSSION

Frozen and fresh

When testing the difference in receptor expression between fresh samples and those which had undergone cryopreservation, expression levels of BCMA and BAFF-R were concordant between fresh and frozen samples and studies have too supported that there are no significant alterations to signalling detected by flow cytometry . Thus the use of frozen samples was considered appropriate, considering the rarity of JSLE and the limited fresh samples available. In regards to TACI there appeared to be an alteration in expression levels of the frozen sample to that of fresh, and alternative studies have too reported significant changes in frozen PBMCs It is therefore important that we take caution when interpreting our results for TACI.

Receptor expression on B cells (CD19+)

It was found that on B cells, JSLE patients had higher BAFF-R, TACI and BCMA expression levels than of controls and JIA, and of the receptors BAFF-R was the most highly expressed on B cells, although none of which was of statistical significance. This increase in JSLE receptor expression may well be an appropriate reflection of the elevated circulating levels of circulating BAFF in our JSLE patients. As previously shown (figure 2), BAFF-R and BCR signalling cooperate to enable auto reactive immature B cells to escape negative selection and join the naive B cell repertoire. Moreover, studies have confirmed that BAFF-R is upregulated following BCR ligation Thus, it could be postulated that elevated BAFF-R expression may represent an elevated degree of BCR ligation, and leave us to question whether there are additional factors, independent of the actions of excess BAFF, which can cause B cell hyper reactivity in JSLE.

It may appear reasonable that BAFF-R was the most highly expressed of the receptors; literature has claimed BAFF-R to be the main receptor mediating BAFF signals for B cell survival and maturation (Thompson,J), with normal B cell development in TACI and BCMA deficient mice( get taci def ref) BAFF-R is the predominant receptor expressed on B cells, being widely expressed on all B cells except for bone marrow plasma cells (with taci n bcma more restricted ?) BAFF-R could therefore be deemed as the main receptor of focus when studying the BAFF signalling system with regards to autoantibody production and disease manifestation. Considering this, our results may demonstrate that besides elevated serum levels of BAFF, altered BAFF-R expression is an additional contributing factor to abnormal B cell homeostasis, and thus the immunopathogenesis of JSLE.

In contrast to our study, former studies investigating BAFF receptor expression in SLE demonstrated that total receptor expression was in fact decreased or unchanged compared to healthy controls. The reduced availability of BAFF-R on B cells is explained by increased occupancy of the receptor, due to the elevated serum levels of BAFF This therefore challenges what may have earlier been proposed by this study. It is however important to clarify that the elevated receptor expression which has been noted in this study is not of statistical significance. Nonetheless, expression also certainly does not appear to be lower or alike to healthy controls. Undoubtedly these disparities found to SLE studies requires further investigation, but could be a possible reflection of an altered disease profile in JSLE, associated with the generally increased disease severity exhibited in children and their phenotypic differences from adults with SLE Also, a simpler explanation could be that fewer receptors on our JSLE samples were occupied due to low circulating BAFF levels. Seeing as levels of BAFF have been correlated with disease activity and all of the JSLE patients were on treatments ultimately designed to control disease activity.

In some aspects, JIA and JSLE are very similar in that a loss of tolerance and autoantibody production underlie the key role of B cells in development of disease Interestingly, our JIA inflammatory control group had and alternate and generally lower receptor expression profile to that of our JSLE. Despite studies on JIA displaying elevated blood leukocyte BAFF mRNA levels, plasma BAFF levels are reported normal in JIA (hong). This may explain the dissimilarity in receptor expression profile seen to that JSLE. This also highlights that the cellular mechanisms instigating JSLE pathogenesis are unique to other autoimmune disorders.

Receptor expression on CD19- PBMCs

It was interesting to find that the receptors for the ‘B –cell activating factor’ were also detected on non-B cells of the PBMC population. JSLE patients expressed statistically higher BCMA and BAFF-R than controls and TACI expression also appeared higher. However, BCMA was the most highly expressed receptor on CD19- cells of all three patient groups and expression exceeded that detected on B cells. BCMA has been found to have a much more restricted pattern, being found only on antibody producing plasma cells and plasmablasts, supposedly dispensable for early B-cell differentiation and instead involved in the maintenance of long lived plasma cells This signifies that the BAFF receptors have specific roles with different primary functions which ultimately effect B cell homeostasis in different ways. CD19 is a marker for B cells from the earliest recognisable lineage, but expression is lost during maturation into a plasma cell. Therefore, the high levels of BCMA expression detected may thus be a possible reflection of predominant BCMA expression on plasma cells which is not detected by the anti-CD19 mAb.

Receptor expression on T cells

The CD19- cells include T lymphocytes monocytes and dendritic cells. It seemed plausible to investigate T lymphocytes, considering a former study had noted BAFF-R expression on some T cells. Despite successful detection of receptors, no trends were found on T cells; JSLE patients did not have the highest expression for any of the BAFF receptors and there appeared to be lots of variability and inconsistency in expression patterns between receptors and patient groups. It seemed somewhat abnormal to find relatively prominent BCMA expression on T cells , as this is contradictory to other studies claiming that BCMA is almost exclusively expressed on antibody producing cells . A possible explanation for these differences may be that this specific experiment has not have been investigated before as there are a limited number of studies that have monitored BAFF receptor expression on JSLE cell types using flow cytometery. Many studies on BAFF receptors has been conducted on healthy subjects and in support our results do demonstrate that healthy patients have exceptionally very BCMA expression. Nonetheless this is a topic that may require further investigation in the future, with specific attention on SLE patients.

On both CD19+ and CD19- cells, TACI was significantly the least expressed receptor observed across all patients groups. Emerging reports have suggested TACI to have a negative regulatory on B cell homeostasis and autoimmunity. TACI deficient mice have an increased number of mature B cells and increased levels of serum immunoglobulin . As a result of this role, TACI expression may be more tightly regulated and restricted, as exemplified in our results. TACI receptor expression on T cells was more varied across different patients groups; with control patients expressing notably more TACI. From this it may be postulated that healthy subjects bearing fewer autoreactive B cells, may do so due to stronger negative regulation of B cell survival signals on T cells.

Nonetheless other literature has shown evidence for a positive regulatory role, implicating a cooperative role between TACI and Toll like receptors (TLRs) to produce pro-inflammatory autoantibodies. TLRs up regulate TACI and literature has claimed TLRs to be involved in the pathogenesis of SLE ; explaining that ‘TLR signalling promotes BAFF effects through TACI’. Therefore, TACI appears to play a divergent role and care must be taken when interpreting results as its expression may be potentiated by multiple factors and its role in B cell homeostasis still remains elusive.

When briefly looking at Neutrophils, BAFF-R and BCMA expression appeared significantly low and thus, although these cells may produce BAFF, it cannot be confirmed that neutrophils express receptors for BAFF. The results for TACI however may implicate that there is some TACI expression on neutrophils. To date, no literature has implicated TACI expression on neutrophils. However, considering the modest number of samples tested and the temperamental behaviour of the anti-TACI mAb, as seen on fresh and frozen samples, no confident conclusions can be established. One possible explanation for detection of TACI on neutrophils is that the fluorescence measured from the anti-TACI mAb may be due to non-specific binding.

Literature has confirmed that BAFF does not solely affect B cells and our results appear to support this. BAFF signalling is highly interconnected with other features of the immune system and appears to have a variety of alternate roles other than B cell survival which require further elucidation. Nonetheless, this insinuates that therapies antagonising BAFF will also affect the homeostasis of other cells which may cause some unwanted side effects. This further highlights the importance of understanding complex signal system that BAFF and its receptors mediate to discover and develop innovative therapies for improving the quality of life for JSLE sufferers. Former research on SLE expression of the BAFF receptors, have reported a down regulation in expression in SLE, and significantly the degree of receptor reduction has been correlated with changes in disease activity.Therefore measuring receptor expression could act a novel disease activity biomarker in SLE and other autoimmune diseases such as Sjögren's syndrome and rheumatoid arthritis, where elevated serum BAFF levels also contributes in their pathogenesis

The most significant challenge posed in studying the immunopathogenesis of JSLE is the unique variability of the disease, its complex pathological process results in a diverse range of afflictions and manifestations. Consistent with this, the complexity of the immune system too provides potential for the impairment of a variety of features to cause one disease, thus the BAFF signalling may not be the main force driving the pathogenesis in all patients. Monitoring BAFF-R expression could be particularly useful for SLE treatments plans, for example in subsets of patients that do not seem to benefit from Belimumab. Testing BAFF-R expression following diagnosis can be a useful and innovative biomarker to recognize whether BAFF antagonism will be an effective therapeutic approach. This could be economically beneficial considering the expensive cost of Belimumab and allows tailored therapy for patients.

Limitation of the study:

There are a number of limitations of the research conducted which should be considered alongside the results found and conclusions reached. Firstly, time was a major limitation and considering the variability in the disease activity and treatment course of the JSLE and JIA patients, the sample size of the main study may not be an adequate representative of the population and trends observed. Of the work conducted on T cells and Neutrophils, the sample size is significantly too small to deduce any valid assumptions or conclusions. With more time this investigation could be improved by increasing the sample size for all experiments conducted, particularly on T cells and Neutrophils, this would allow us to make better judgements on abnormal results and would give results greater statistical power and verify any deliberations discussed.

Furthermore all JSLE and JIA patients were on a variety of drug therapies, these immunotherapies affect the immune system and are used to control the disease. It would therefore be injudicious to consider that a ‘manipulated’ immune system will not affect what this study is investigating. This can exemplified in our study, by one JSLE patient on Rituximab treatment, whose results were removed from the data because treatment had caused depletion of the B lymphocyte population.

Furthermore, The JSLE group studied all varied in disease activity, correlations have been drawn between disease activity and BAFF and BAFF-R expression, and this may consequently impact analysis of results and distinctions that can be made about JSLE receptors expression profiles. There was also considerable variation in the range of receptor expression amongst the JIA group. The JIA group included patients with three different types of the disease, which vary in severity. Some JIA subgroups share stronger similarities to JSLE; the presence of anti-nuclear antibodies (ANA) is also a hallmark in the Oligoarticular subgroup of JIA patients .Thus in future, using solely oligoarticular samples may be beneficial in distinguishing in which aspects the pathogenesis of JSLE may be unique to that of a similar disease and will also provide more consistency in results as an inflammatory control group.

Upon testing receptor expression on fresh and frozen cell samples the inconsistency in expression of TACI from the same sample raised a concern and questions the reliability and accuracy of the data obtained for TACI. TACI also had a typical expression on non B cells such a neutrophils. Additionally, some concerns were raised about the levels of BCMA expression; some data appeared abnormally high and deviated from typical values, there also appeared to be significant BCMA expression on T cells which literature does not support. Considering this, caution must be taken when interpreting our results for TACI and BCMA as reliability of the data may be compromised.

Next steps could include monitoring differences in receptor expression on other cells, such as monocytes, which reportedly also express receptors for BAFF. Studying the expression profiles of the BAFF receptors on these cell types could enhance our knowledge on the diverse roles of the BAFF signalling in the immune system.

This study measured BAFF receptor expression however this does not give information on the quantity of receptors which were occupied by BAFF. As revealed in our discussion, there were some disparities identified between conclusions drawn from research on receptor expression of SLE patients and from this study. This investigation could be taken further by conducting tests looking at receptor occupancy and the persistence of occupancy. This may facilitate our understanding of the causes for the differences in trends noted in this study compared to others and provide a more direct implication of how BAFF signalling is altered in JSLE.

From a new perspective, measuring signalling events downstream of the BAFF receptors which mediate the functions of BAFF could be a valuable method for understanding abnormalities in BAFF signalling activity In JSLE. Studies show that TACI and BCMA signal via the classical NF-kB pathway , whereas BAFF-R can also induce stimulation of the alternative NF-kB pathway (Claudio et al., 2002). Thus a further research direction could involve measuring and comparing the activity of both the NF-κB signalling pathways in JSLE patients and in health. The fact that two distinct pathways are involved may also allow us to deduce which of the receptors are predominantly employed, to mediate survival related functions. This provides an alternative method than measuring receptor expression as a means of understanding abnormalities in BAFF signalling contributing to the immunopathogenesis of JSLE.

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