HOMOGENITY IN TH9 CULTURESDaniel Chris Gomes, Benjamin Joseph Ulrich and Mark H. Kaplan

Department of Pediatrics, H.B. Wells Center for Pediatric Research, Indiana School of Medicine, Indianapolis, IN, USA

ABSTRACTCD4 T helper cells regulate inflammation that is critical for immunity to pathogens but also lead to autoimmune disease and allergies. Among the T-Helper subsets, the IL-9-secreting population of Th9 cells mediates anti-tumor and provide a powerful mechanism for immunity against helminth infections. Moreover, IL-9 plays an important role in Inflammatory Bowel disease and a critical role in murine models of asthma and food allergy. When Th9 cells are cultured in vitro from CD4+ naïve T cells, one might assume the population to be homogenous. However, a culture of TH9 cells contains both IL-9-secreting and non-secreting cells. We hypothesized that the Th9 genetic program is restricted to the IL-9-secreting population. We examined the transcriptional signature of IL-9 producing Th9 cells, using an IL9 reporter mouse that expresses Citrine Yellow Fluorescent Protein from the Il9 locus. Selecting genes that are preferentially expressed in the Th9 subsets we compared relative expression in sorted Citrine positive and negative populations after five days of culturing in Th9 conditions. We observed that Il9 and a subset of Th9 enriched genes are enriched within the Citrine-positive population. However, many of the genes were similarly expressed in both populations. Overall, our data demonstrated that many of the genes associated with the Th9 signature are expressed in both IL-9-secreting and non-secreting populations within a Th9 culture, and suggest that heterogeneity within a TH9 population cannot be defined strictly by IL-9 expression. Future studies will define the molecular basis for this heterogeneity.

INTRODUCTION

MATERIALS & METHODS

RESULTS

Figure 2: T-Helper cell subsets and their differentiation conditions. Modified from Kaplan M.H., Hufford M.M., Olson M.R.Nature Reviews Immunology2015, May;15(5):295-307.

CONCLUSIONS

At times in science we assume in-vitro cell cultures consist of homogenous populations. However, what we actually have in culture are a number of populations with a considerable amount of heterogeneity. Therefore, it is important to ask the question in the case in-vitro Th9 cultures whether the TH9 genetic program traditionally considered is restricted to cells that are secreting IL-9. Standard TH9 conditions, which includes TGFβ and IL-4 can produce IL-9 secreting TH9 cells developing from naïve CD4+ T cells (Fig. 2).

Previously published (1), microarray analysis from standard TH9 cultures outlined a TH9 genetic program which lists a number of genes that have been upregulated in TH9 cells. Starting from this microarray analysis and additionally RNA-seq data available publicly we found additional genes that were preferentially regulated in TH9 cultures (Fig.3). Focusing on five genes based upon the expression ratio between TH9 populations compared to TH2 populations, we asked the question whether the expression of these genes are restricted to cells that are secreting IL-9.

Figure 3: Microarray analysis of Th9 genetic program

Figure 4. Experiment 1: In-vitro culture of T-Helper cells to be used in gene expression RT-

PCR experiment.

Figure 5a: IL9 Citrine Reporter mouse gene.Figure 5b: Dot plot of a flow cytometer analysis of Citrine versus IL9 populations.

a b

Figure 6: Experiment 2: In-vitro culture of TH9 and TH2 cells and IL9 detection

Figure 7: RT-PCR data of gene expression in TH1, TH2, TH9 and TH17 cultures. Most of these genes which include Erg, Il17rb, IL9, Rab38, Irf4, Pcdh17 and Il1rl1 are preferentially expressed in the TH9 subset except for Lrrc4 and Ear10 which shows almost similar amounts of preferential expression in TH9 and TH17 cultures.

Figure 8: IL9 gene expression confirmed the effectiveness of the reporter. Citrine is a marker for IL9. Which is why a significant increase in expression of IL9 in the Citrine positive population can be observed. The data for Pcdh17 and Il1rl1 shows that these genes are preferentially expressed in Citrine positive populations. Batf, Irf4, Erg, Rab38, Ccr8, Itgae, Il17rb, Il1rn, Ahr shows preferential expression in both TH9 Citrine positive and negative populations.

Figure 9: The Th9 genetic program is largely intact in Citrine negative cells

As mentioned earlier, we have examined genes traditionally considered to be a part the of the TH9 signature expressed specially in the TH9 subset. Since TH9 cells are defined through their expression of IL-9, these genes were thought to be enriched in the IL-9 producing population (Citrine positive population). Interestingly we found the opposite result with several of these genes expressed at similar levels in non-IL-9 producing populations (Citrine negative population). After going back to the microarray data we discovered another list of genes upregulated in TH9 cells and found that the genes Il1rl1 and Pcdh17 were enriched at similar levels to IL-9 in the IL-9 producing populations. This demonstrates that some genes are linked to IL-9 expression in TH9 cultures. The discovery of what common factor that regulates these genes linked to IL-9 that is different from the genes traditionally upregulated in TH9 cultures will provide a breakthrough in defining the true TH9 gene signature.

ACKNOWLEDGEMENTSI acknowledge the Herman B Wells Center for Pediatric Research and Earlham College Center for Integrated Learning for summer support and supplies. I would like to especially thank my talented and enthusiastic Kaplan Lab researchers Mark H Kaplan, Ph.D. Benjamin Joseph Ulrich, Matthew Olson, Ph.D., Rukhsana Jabeen, Ph.D. , Nahid Akhtar. The support of my PI, Dr. Mark H. Kaplan and my supervisor Benjamin Joseph Ulrich, throughout the different stages of this project is very much appreciated.

ADDITIONAL REFERENCES(1) Jabeen R, Goswami R, Awe O, Kulkarni A, Nguyen ET, Attenasio A, Walsh D, Olson MR, Kim MH, Tepper RS, Sun J, Kim CH, Taparowsky EJ, Zhou B, Kaplan MH, Th9 cell development requires a BATF-regulated transcriptional network, J Clin Invest. 2013 Nov;123(11):4641-53.(2) Mark H. Kaplan, Matthew M. Hufford and Matthew R. Olson, The development and in vivo function of T helper 9 cells, Nature reviews Immunology, Vol. 15, pg. 295-305, May 2015.

RESULTS (continued)For our experiments, two sets of RT-PCR experiments were performed with these genes. The first set included the expressions of these genes in TH1, TH2, TH9 and TH17 cultures. The spleen was first harvested from the mice. The naïve CD4 positive T cells were then allowed to culture under standard TH1, TH2, TH9 and TH17 conditions. On day 3 the cells were transferred to a bigger plate and allowed to expand. On Day five, one million cells from each condition were harvest to be used in RT PCR to observe the gene expression of the TH9 signature genes.

Our hypothesis for the second part of our experiment was that the TH9 genetic program was restricted to IL9 producing cells only, which is why for this set of experiments we used an IL9 Citrine Reporter mouse. The IL-9 reporter mouse has a Citrine reporter gene in place of the IL9 gene, that replaces IL-9 production with a Yellow Florescent Protein allowing the cell to sort and purify IL-9 secreting cells. The RT-PCR gene expression experiments were then run on the populations that were collected from the sorted cells. Therefore, in this second set of experiments we observed the gene expressions of the selected genes in TH2, TH9 Citrine positive and TH9 Citrine negative populations.

Herman B Wells Summer Interns 2016