Source: UNIV OF NEW MEXICO submitted to NRP
CONTRIBUTIONS OF ORGANIZED NASOPHARYNX-ASSOCIATED LYMPHOID TISSUE TO RAINBOW TROUT IMMUNE RESPONSES TO VACCINATION
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1022609
Grant No.
2020-67015-31457
Cumulative Award Amt.
$500,000.00
Proposal No.
2019-05906
Multistate No.
(N/A)
Project Start Date
Jun 1, 2020
Project End Date
May 31, 2024
Grant Year
2020
Program Code
[A1221]- Animal Health and Production and Animal Products: Animal Health and Disease
Recipient Organization
UNIV OF NEW MEXICO
(N/A)
ALBUQUERQUE,NM 87131
Performing Department
Biology Department
Non Technical Summary
Fish farming provides all US consumers with a high-quality food source. Intensive fish farming, however, can result in disease outbreaks. Most of the diseases affecting farmed fish start at mucosal barriers such as the skin, gills, gut and olfactory organ. Thus, there is a need for effective mucosal vaccines for use in aquaculture. Nasal vaccination is highly efficacious and protects salmonids from viral and bacterial pathogens. We hypothesize that not only the olfactory mucosa, but also immune structures recently identified in other areas of the trout nasal cavity, are involved in the immune response during vaccination. The goal of this proposal is to characterize the unique aspects of these previously ignored structures of the rainbow trout nasal cavity. To that end, we will evaluate the innate and adaptive immune responses in rainbow trout during the course of vaccination using viral and bacterial vaccines delivered intranasally or by injection. We predict that these structures play complementary immunological functions to those in the olfactory mucosal epithelium. These findings will increase our understanding of how fish defend themselves from mucosal infections and will provide a platform to design better mucosal vaccines for farmed salmonids. These improved vaccines will increase the sustainability and production of the fish farming industry in the US.
Animal Health Component
35%
Research Effort Categories
Basic
65%
Applied
35%
Developmental
0%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
31508101090100%
Knowledge Area
315 - Animal Welfare/Well-Being and Protection;

Subject Of Investigation
0810 - Finfish;

Field Of Science
1090 - Immunology;
Goals / Objectives
The long-term goal of this proposal is to understand the full potential of nasal vaccines for use in aquaculture. We have shown that nasal vaccination with either bacterial or viral vaccines inducespotentlocal and systemic innate immune responses. We also determined that nasal vaccination confers longer protection than injection vaccination when using a live attenuated viral vaccine but not a bacterin. We recently noted the presence of a lymphoid structure that we have coined as organized NALT(O-NALT) in the nasal cavity of trout. This previously overlooked structure is rich in lymphocytes but its role in the fish mucosal immune system is unknown. Thus, this new finding raises the question: what is the contribution of O-NALT to fish immunity and to the innate and adaptive immune responses during vaccination? The objectives of this proposal are 1) To perform a detailed molecular and cellular characterization of trout O-NALT; 2) To analyze the innate and adaptive immune responses in O-NALT in rainbow trout in response to vaccination; 3)To evaluate the B and T cell repertoire responses in trout O-NALT in response to vaccination.
Project Methods
Aim #1: Molecular and cellular characterization of trout O-NALTThe methods to be used are: Serial sections of whole trout nasal cavity paraffin blocks will be stained with H&E to determine the size and morphology of trout O-NALT in the absence of any infection or vaccination. Using previously collected paraffin blocks of a developmental series of trout larvae ranging from 15-degree days (DD) to 600 DD, we will establish the earliest developmental stage at which O-NALT can be observed in troutby light microscopy. O-NALT ultrastructure will be characterized by transmission electron microscopy. Control nasal cavity cryosections will be stained with the following trout specific antibodies: MHC-II, pIgR, IgM, IgT, IgD, CD4-1, CD4-2, CD8,TCRa, TCRd and activation-induced cytidine deaminase (AID). All these antibodies have been kindly provided by Dr. O. Sunyer. Total RNA from LCM-captured O-NALT and d-NALT will be used for transcriptomic analyses using Illumina RNA-Seq.Aim #2: Analysis of innate and adaptive immune responses in trout O-NALT to vaccination.Methodology: Rainbow trout (20g) will be obtained from Lisboa Springs Hatchery, Pecos, New Mexico and vaccinated with ERM, IHNV or mock-vaccinated. Vaccines will be delivered I.N or i.p/i.m as previously described [11]. To capture innate immune responses, we will collect samples at 1, 3 and 7 days post-vaccination. To capture adaptive immune responses, trout will be sampled at 1, 3 and 6 months post-vaccination. 24 h prior to sampling, trout will be injected intravenously with Edu as previously described. Serum samples will be collected at each time point for use in Aim#3. This will allow for identification of proliferating cells by IF microscopy in d-NALT, O-NALT and spleen. Whole nasal cavity samples will be dissected and used for: routine histology and H&E staining, transmission electron microscopy, cryosections followed by IF staining, cryosections followed by LCM for RNA-Seq. N=6 per time point and per experimental group will be sampled for each of the readouts listed. Light microscopy and TEM will be used to characterize size, morphological and ultrastructural changes in O-NALT and d-NALT over time. O-NALT size will be normalized to the size of the fish as explained in Aas, 2014. Cellular composition of O-NALT and d-NALT will be determined by IF microscopy using trout specific antibodies: MHC-II, pIgR, IgM, IgT, IgD, CD4-1, CD4-2, CD8,TCRdand AID. Co-localization of AID expression in B cells will suggest active somatic hypermutation (SHM) of the BCR. Total proliferation levels will be quantified by counting Edu+ cells. Identification of proliferating cells will be done by double staining (Edu+and specific cell marker). Based on these data, we will select the most relevant timepoints for RNA-Seq studies. RNA-Seq will be performed as described in Aim#1 using mock-vaccinated fish as the control.Aim #3:Evaluation of the Band T cell repertoire responses in O-NALT in rainbow trout in response to vaccination.Methodology: NGSmethods have been applied to study immune repertoires of whole organisms or specific tissues. In this aim, we propose to apply NGS to establish the differences in the B and T cell repertoires of trout O-NALT, d-NALT and spleen using the ERM and IHNV vaccine models. Homozygous rainbow trout (Swanson line, mean weight 20g) will be obtained from Dr. Gary Thorgaard's laboratory as it was done in our current award.Trout will be divided into fivedifferent experimental groups: mock vaccinated, I.NERM vaccinated, i.p ERM vaccinated, I.N vaccinated with IHNV vaccine, i.m.vaccinated with IHNV vaccine. Fish (N=8) will be sampled 1, 3, and 6 monthspost-vaccination. Additionally, outbred trout from Lisboa Springs Hatchery (20g) will be also vaccinated using the same experimental set-up and sampled at 1 month after vaccination since we know that at that time, repertoire perturbations can be detected already in d-NALT. Serum samples will be collected at each time point to measure specific IgM and IgT titers as recently described. Total RNA will be extracted O-NALT, d-NALT and spleen. O-NALT and d-NALT RNA will be obtained from LCM-captured serial cryosections. RNA samples will beconverted to 5'RACE ready cDNA (SMARTer RACE cDNA amplification kit, Clontech). The 5'RACE cDNA will be prepared using poly-dT to get all poly-A transcripts or specific primers for IgM, IgT and TCR constant domains. Amplicons will be produced using nested-PCRas recently described by us. Primers containing Illumina MiSeq adaptor /index /unique barcode labels, with IgM, IgT, TCRa and TCRd chain specific primers will be used to amplify the repertoire of interest.

Progress 06/01/20 to 05/08/24

Outputs
Target Audience:Our target audience has been aquatic animal health specialists, fish immunologists, aquaculture researchers andvaccinologists as well as aquaculture vaccine companies Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Under this award a graduate student (Ben Garcia) has been trained for three years in fish vaccinology, mucosal immunity, fish immunology and bioinformatic analysis of repertoires. Ben has already advanced candidacy towards his PhD.Additonally, another graduate student has been trained in the same areas for a year.We have also provided training opportunities for several undergraduate students who has been trained in fish health, fish care, anatomy, tissue collection, microscopy etc. In terms of professional development for Irene Salinas: She was promoted to Full Professor in 2022. She joined the Board of the International Society forFish and Shellfish Immunology in 2023 and she became the lead of their DEI Chapter. She also became the President Elect of the International Society for Developmental and Comparative Immunology in September 2023. She received the UNM Creative Research and Scholarship award in 2023 for her work on rainbow trout vaccines and nasal immunity. How have the results been disseminated to communities of interest?We have been very active at disseminating our work in different ways. Irene Salinas has visited vaccine companies (Zoetis and Hipra) and given seminars on nasal vaccines and O-NALT. Irene Salinas has given many plenary talks and invited seminar at important venues such as the International Conference for Aquatic Animal Health in Chile, 2022, BioAqua, Cuba, 2022, International Society for Fish and Shellfish Immunology Congress (Bodo, Norway, 2022), International Society for Developmental and Comparative Immunology Congress (Wageningen, Netherlands, 2023) etc. Graduate students from the laboratory have also presented the findings derived from this award at several conferences including the UNM Biology Research Days (two talks, one poster), the American Associatiopn of Immunology Conference (Poster) in May 2024, Chicago, and also in the American Fisheries Society Fish Health Section Seminar series. We have also disseminated our findings to the scientific community via our peer reviewed publications. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? We have accomplished the majority of our proposed goals. We completed all experiments proposed in Aims 1 and 2. We are currently writing the manuscript pertaining our findings on myeloid cells in the trout O-NALT. We have discovered presence of macrophages, dendritic cells and granulocytes in the O-NALT. We also identified MHC-II expression patterns in this lymphoid organ and how it develops during ontogeny. We mapped the ontogeny of the O-NALT in great detail and determined that it appears around 1400 degree days in our trout (maintained at 16C). We have also characterized in detail the B and T cells that are present in trout O-NALT at the steady state and following prime and boost intranasal vaccination. We showed that IgM B cells specifically proliferate in the O-NALT and they also undergo apoptosis, indicating that this site is likely a site for B cell selection in the trout mucosa. We also detailed the expression of criticalmolecular markers (chemokines, cytokines, bcl-2, bcl-6, aicda) that are reponsible for the germinal center reaction in mammals in the trout O-NALT before and after vaccination. Finally, we have just finalized the first B cell repertoire analysis in trout O-NALT in response to intranasal vaccination and we are currently analyzing the data.

Publications

  • Type: Book Chapters Status: Published Year Published: 2021 Citation: Mucosal Immunity in Fish in Principles of Fish Immunology from Cells and Molecules to Host Protection
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Garcia B, Dong F, et al. A novel organized nasopharynx-associated lymphoid tissue in teleosts that expresses molecular markers characteristic of mammalian germinal centers. The Journal of Immunology 209: 2215-2226.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: The ontogeny of the organized nasopharynx associated lymphoid tissue in rainbow trout (Onchorhynchus mykiss). Developmental and Comparative Immunology 148, 104950
  • Type: Conference Papers and Presentations Status: Awaiting Publication Year Published: 2024 Citation: B cell receptor repertoire analysis of of the organized nasopharynx-associated lymphoid tissue (O-NALT) following prime and boost intranasal vaccination in rainbow trout
  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Benjamin J Garcia, Alexis Reyes, Chrysler Martinez, Yago Serra dos Santos, Irene Salinas. Ontogeny of the organized nasopharynx-associated lymphoid tissue in rainbow trout. bioRxiv 2023.08. 04.552019
  • Type: Journal Articles Status: Other Year Published: 2024 Citation: Garcia B, AbouIsmail A, Musayeva N, Salinas I. Characterization of antigen up taking cells and antigen retention in the organized nasopharynx-associated lymphoid tissue of rainbow trout
  • Type: Journal Articles Status: Other Year Published: 2024 Citation: Garcia B, Magadan S, Salinas I. IgM B cell repertoire in the rainbow trout is modified upon intranasal vaccination in an antigen-dependent manner


Progress 06/01/22 to 05/31/23

Outputs
Target Audience:The target audiences for this reporting period have been: -Industry: I have been in direct converations with several indsutry partners who are world leaders in fish vaccine products. Interactions occured in person at the ISAAH and ISFSI meetings as well as via invited in person visits to Hipra inGirona, Spain in July 2022 andPharmaq Norwayin December 2022. -Fish health experts, fish immunologists and fish vaccinologists worldwide: I have presented our work in the International Society for Aquatic Animal Health Meeting (ISAAH) in Chile in September 2022 as a plenary speaker of the conference as well as the plenary speaker of the International Society for Fish and Shellfish Immunology (ISFSI) in Bodo, Norway, in December 2022. -Broader aquaculture and biotech community: via plenary talk at BioAqua (Cuba, April 2022) and aposter presentationat Aquaculture America Feb 2023 -Broader mucosal immunology scientific community: I was a plenary speaker at the Society for Mucosal Immunology symposium held at theAmerican Association of Immunology congress in Portland, Oregon in May 2022 and presented our USDA work. -General public: via press releases (https://advance.unm.edu/unm-biologists-make-breakthrough-in-fish-mucosal-immune-system-research/) of our papers as well as social media (@DrSalinasLab) Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Thanks to this award, three graduate students have received training in fish immunology, vaccinology and aquaculture. Alexis Garcia, a black latina is currently working as an undergraduate student lab assistant in the project. Ben Garcia presented the work at NACIW 2022 in BANFF and won the first award for the best student oral presentation. Ben Garcia also presented a poster at the University of New Mexico Research Days and won the first award for graduate student poster presentation. How have the results been disseminated to communities of interest?As explained before we have been very active during this period to disseminate our work to all stakeholders via -Peer review papers - Conference talks and posters: BioAqua 2022, AAI 2022, North American Comparative Immunology Workshop 2022, International Society for Aquatic Animal Health (Santiago de Chile, 2022), International Fish and Shellfish Immunology (Bodo, Norway, 2022), Aquaculture America (New Orleans, Feb 2023) -Invited talks (PI: Irene Salinas) - Visits to fish vaccine companies: Hipra and Pharmaq - Press releases - Social media (@DrSalinasLab) What do you plan to do during the next reporting period to accomplish the goals?We are finishing the quantification of the different innate and adaptive immune cell types during development (the plan is to have it finished in the next two months and write the manuscript to DCI). Meanwhile we need to sequence the repertoire libraries (they ar ready to be submitted) and analyze the data.

Impacts
What was accomplished under these goals? We have completed most of our objectives in Aim 1 and 2 and collected all the samples for repertoire analyses in Aim 3. We are currently producing the libraries for repertoire sequencing and we will devote the rest of the time for data analysis and manuscript preparation. We are also finalizing the ontogeny part of the project, we finished the time series sample collection, we have performed H&E staining of all developmental points in different orientations and determined that the appearance of a primordial O-NALT occurs earlier than what we thought. Our first observation is now at 529 degree days. Our major findings regarding the adaptive immune response in O-NALT in response toprime and boost nasal vaccination are groundbreaking both from a basic and applied point of view (Garcia et al., 2022 The Journal of Immunology). Our work has revealed that fish do have lymphoid structures at mucosal sites such as the nasal cavity and that these structure express molecular markers that make them candidates for mammalian germinal centers. This means that fish mucosal vaccines must target these lymphoid structures where B cells are likely being selected during the process of affinity maturation. Importantly, these structures and the antibody maturation process that occurs within these structuresmust be considered when fish vaccine regimes are optimized. Our manuscript was selected as the top read of the month last December at the Journal of Immunology and our image made it as the cover of the issue.

Publications

  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Benjamin Garcia, Fen Dong, Elisa Casadei, Julien Ress�guier, Jie Ma, Kenneth D. Cain, Pedro A. Castrillo, Zhen Xu, Irene Salinas; A Novel Organized Nasopharynx-Associated Lymphoid Tissue in Teleosts That Expresses Molecular Markers Characteristic of Mammalian Germinal Centers. J Immunol 1 December 2022; 209 (11): 22152226. https://doi.org/10.4049/jimmunol.2200396
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2022 Citation: Irene Salinas, Benjamin Garcia, Fen Dong, Elisa Casadei; Discovery of an organized nasopharynx-associated lymphoid tissue in the nasal cavity of rainbow trout and its role in secondary adaptive immune responses to nasal vaccines. J Immunol 1 May 2022; 208 (1_Supplement): 124.19. https://doi.org/10.4049/jimmunol.208.Supp.124.19
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2022 Citation: Garcia B, Fen Dong, Elisa Casadei, Julien Ress�guier, Jie Ma, Kenneth D. Cain, Pedro A. Castrillo, Zhen Xu, Irene Salinas. Oral presentation. North American Comparative Immunology Meeting BANFF, Canada, June 2022
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2022 Citation: Irene Salinas. Teleost nasal immunity and its application to aquaculture vaccines. BioAqua, Varadero, Cuba, April 2022.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2023 Citation: The ontogeny of organized nasopharynx associated lymphoid tissue in rainbow trout: implications for vaccination. Poster. Aquaculture America 2023, New Orleans, February 2023, USA. Elisa Casadei, Alexis Reyes, Ben Garcia, Fen Dong, Irene Salinas


Progress 06/01/21 to 05/31/22

Outputs
Target Audience:We have targeted the general public via social media (@DrSalinasLab) We have made efforts to communicate our results to the Aquaculture community in the conference BioAqua, Cuba, APril 2022 We have communicated findings to veterinary immunologists, comparative immunologists and general immunologists and vaccinologists via invited seminars and conference presentations including a poster at the American Association of Immunologists (Portland, Oregon, May 2022), an invited conference presentation for the Society for Mucosal Immunology at AAI Portland, May 2022; an invited presentation at the Food and Allergy Science Initiative at the Broad Institute (Boston, MA, June 2 2022), an invited seminar at the Harvard Medical School Systems Biology Department in May 2022. Finally, the entire scientific community will be reached via our submitted paper Garcia et al., which is under review at the Journal of Immunology right now. The wider Fish Disease and Aquaculture community will be reached next September in Chile at the International Aquatic Animal Health Conference. Changes/Problems:We performed a preliminary repertoire sequencing round using RNA from LCM captured O-NALT and we obtained not optimal read numbers (hundreds to low thousands). Thus, using this approach, we will not obtain good enough sequencing depth for quality repertoire analyses. Based on this and our finding that the entire nasal cavity is a lymphoepithelium, LCM does not seem the best approach to follow and we have modified our method to tissue dissections as explained before. What opportunities for training and professional development has the project provided?Under this award, we established very fruitful collaborations with Dr. Julien Resseguier at the University of Oslo who is an expert in teleost MALT imaging. Under this award, new PhD student in the Salinas laboratory, Benjamin Garcia, has been trained in trout vaccination, nasal immunity, laser capture microdissection, microscopy etc..Additionally, another PhD student,Amir Mani was also partly funded by the award. Amir Mani advanced his candidacy earlier this year. Dr. Salinas was promoted to Full Profesor just recently. How have the results been disseminated to communities of interest?As mentioned before, we have presented our results in several national and international meetings, many invited seminars, via conference posters, and in peer reviewed articles and a book chapter. We have now one more paper to submit with the ontogeny results and one more paper from the repertoire sequencing data which is undergoing. What do you plan to do during the next reporting period to accomplish the goals?We have modified the plan to perform repertoire. Instead of using RNA from LCM captured O-NALT, we have designed a way to dissect the nasal lymphoepithelium and we will sequence the repertoire from this tissue as well as the olfactory rosette (diffuse NALT), and the spleen. We have noted developmental differences in the composition of the O-NALT from larvae to juveniles. Preliminary results indicate that the % of CD8 T cells is very high in 3-5g animals and that it decreases as animals grow to 30g. This is at the expense of CD4 and IgM B cells becoming much more abundant as animals develop. We want to carefully confirm this observation by performing more microscopy stainings and quantifying cell numbers is more individuals to increase our statistical power

Impacts
What was accomplished under these goals? We have completed most of the aims of our proposal. We have determined when O-NALT first develops in trout (yet to be published), we have chracterized at the cellular and molecular level the trout O-NALT environment and compared it to the olfactory mucosa. We have performed vaccination trials and discovered that, as originally hypothesized, the novel O-NALT structure like acts as a site for B cell selection in the teleost nasal mucosa. This conclusion is based on the fact that IgM B cells enter apoptosis and also proliferate in the trout O-NALT in response to nasal vaccination and that aicda is expressed at very high levels in the O-NALT compared to other regions. We also identified molecular markers in the trout O-NALT that are very similar to markers for germinal centers in mammals. Importantly, while performing this work, we noted that the entire nasal cavity not just the O-NALT lymphoid aggregate forms a continouus lymphoepithelium rich in T and B cells. This observation is very exciting and made us modify our approach for the ongoing repertoire sequencing experiments. Finally, we performed the first prime and boost nasal vaccination experiments and noted that following booster vaccination, B and T cells appear to leave the O-NALT suggesting seeding of other mucosal effector sites such as the lamina propria of the nasal epithelium. This exciting possibility will be followed up in future studies/proposals.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Benjamin Garcia, Fen Dong, Elisa Casadei, Julien Resseguier, Jie Ma, Pedro A. Castillo, Zhen Xu, Irene Salinas. Discovery of lymphocyte aggregates in the nasal cavity of rainbow trout and their role in immune responses to nasal vaccines. The Journal of Immunology Suppl. 2022
  • Type: Journal Articles Status: Under Review Year Published: 2022 Citation: Benjamin Garcia, Fen Dong, Elisa Casadei, Julien Resseguier, Kenneth D. Cain, Jie Ma, Pedro A. Castillo, Zhen Xu, Irene Salinas. 2022. A novel organized nasopharynx associated lymphoid tissue (O-NALT) in teleosts in an environment conducive to B selection. The Journal of Immunology, under review
  • Type: Book Chapters Status: Published Year Published: 2022 Citation: Mucosal Immunity in Fish I Salinas, Y Ding, � Fern�ndez-Montero, JO Sunyer. 2022. Principles of Fish Immunology, 387-443
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Mucosal immunoglobulins of teleost fish: A decade of advances I Salinas, � Fern�ndez-Montero, Y Ding, JO Sunyer Developmental & Comparative Immunology 121, 104079


Progress 06/01/20 to 05/31/21

Outputs
Target Audience:Due to the pandemic, we have not been able to present our work in person. However, we have targeted the Fish Disease community via virtual meetings as well as invited seminars (virtually). We presented in May 2020 our forst set of result in the AFS-FHS. Furthermore, Dr. Salinas has given several talks in different national and international venues where she incorporated findings from this award in her seminar. She has been invited as a keynote speaker in September 2021 for the ISAAH in Santiago de Chile and she will also present results there. We are also preparing the first manuscript containing results from this award and it will soon be submitted for publication. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Under this award we have trained one international visiting PhD student, Fen Dong and a graduate student at UNM Benjamin Garcia. How have the results been disseminated to communities of interest?As mentioned earlier, due to covid, we have been very restricted to meetings. However, we are planning on communicating our results much more widely soon and to also submit our first publication in the next few months. What do you plan to do during the next reporting period to accomplish the goals?We are working on writing the first manuscript and completing the experimental work for aim 3.

Impacts
What was accomplished under these goals? We have finalized all the experiments and data collection for aims 1 and 2. Our results are very exciting and have confirmed the presence of large lymphocyte aggregates in the nasal cavity of teleosts that are largely composed of IgM B cells, CD8 T cells and a few IgT B cells. We have also determined at the molecular level that O-NALT structures have much higher expression of the enzyme AID, responsible for somatic hypermutation and therefore maturation of the immune response. Nasal vaccination trials have also revealed unique cellular dynamics in O-NALT compared to the tips of the olfactory lamella. We have also performed the animal experiments necessary to complete aim 3 goals. We need to perform laser capture microdissection in those samples, extract RNA and prepare libraries for repertoire analyses.

Publications