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SUMMARY STATEMENT 

PROGRAM CONTACT: ( Privileged Communication ) Release Date: 03/04/2011 

Application Number: 1 R21 AI095831-01 

Principal Investigator 

PETRIE, HOWARD T. PHD 

Applicant Organization: SCRIPPS FLORIDA 

Review Group: CMIB 

Cellular and Molecular Immunology - B Study Section 

Meeting Date: 02/03/2011 RFA/PA: PA10-069 

Council: MAY 2011 PCC: I2H 

Requested Start: 07/01/2011 

Dual IC(s): HD, HL 

Project Title: Lymphoid signals for stromal growth and organization in the thymus. 

SRG Action: Impact/Priority Score: 

Human Subjects: 10-No human subjects involved

Animal Subjects: 30-Vertebrate animals involved - no SRG concerns noted 

Project Direct Costs Estimated 

Year Requested Total Cost 

1 

2 

TOTAL 

ADMINISTRATIVE BUDGET NOTE: The budget shown is the requested budget and has not been 

adjusted to reflect any recommendations made by reviewers. If an award is planned, the costs will be 

calculated by Institute grants management staff based on the recommendations outlined below in the 

COMMITTEE BUDGET RECOMMENDATIONS section. 

Text from the application is copyrighted. The awardee provided express permission for NIAID to post the

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Contact Information. Email NIAID's Office of Knowledge and Educational Resources at deaweb@niaid.nih.gov.
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

1 R21 AI095831-01 2 CMIB 

PETRIE, H 

1R21AI095831-01 PETRIE, HOWARD 

RESUME AND SUMMARY OF DISCUSSION: This is a new Exploratory/Development application from 

a very creative and outstanding leader who has made seminal contributions to the field of T cell biology. 

The application proposes to study lymphoid signals that stimulate stromal growth and organization of 

the thymus. The intercommunication between thymocytes and thymic epithelial cells has been a long 

difficult journey although this investigator is well poised to accomplish significant progress. The 

strengths of this application are the outstanding investigator, highly innovative and well written 

experimental plan, state-of-the-art techniques (i.e. microarray-based gene profiling), and the very high 

significance of more fully understanding the correlation between the thymocytes and the other cells in 

the thymus. While this is an ambitious application which is not without risk, this is certainly well within 

this investigator’s capabilities. Thus the overall impact is extremely high and likely to significantly 

advance the field. 

DESCRIPTION (provided by applicant): Like all blood cells, T lymphocytes are constantly lost during 

life, and must be continuously replaced. The thymus is the primary site of de novo T lymphopoiesis. 

Microenvironmental conditions unique to the thymus induce a complex series of developmental events 

in multipotent, marrow-derived progenitors, including positive and negative control of proliferation, T 

lineage specification, functional T lineage asymmetry, self-restriction, self-tolerance, and cell 

death/survival signals. Significant progress has been made in understanding the signals the thymus 

provides to lymphoid progenitors to induce and control these events. However, the proliferation, 

differentiation, and/or survival of the non-lymphoid (stromal) components of the thymus are equally 

dependent on lymphoid cells, and the absence of lymphoid cells (e.g., congenital immunodeficiency 

diseases) results in athymia, even though the nascent stromal cells are functionally competent. The 

signals that lymphoid cells provide to induce stromal growth, differentiation, and/or survival are 

completely unknown. In this proposal, we propose to address this process in a comprehensive fashion. 

Using our recently devised method for global characterization of stromal gene expression in situ, we will 

analyze the dynamic response of stromal cells to the presence of lymphoid progenitors in an inducible 

model of thymic growth. We will then identify stromally-expressed genes that encode receptors, and 

thus may respond to the presence of lymphoid cells, with particular emphasis on those that change 

during specific phases of the growth response (induction, log phase, termination, steady-state). We will 

verify the presence of the cognate ligands for these receptors in lymphoid cells, using informatic 

methods as well as manual means of curation. Cognate receptor:ligand pairs will then be prioritized 

using a variety of criteria, including dynamic patterns of expression, known functional relevance 

(growth, differentiation, survival) in other tissues, the availability of existing genetic models, etc. Finally, 

the biological relevance of a few high-priority candidates will be tested, using appropriate stromalreceptor or lymphoid-ligand mutant mouse models. This project is expected to provide novel insights 

into an unexplored area of biology that will not only fuel a better understanding of this process, but will 

facilitate the development of more mechanistic studies to characterize the unique, two-way interactions 

that occur between lymphoid progenitors and stromal cells in the thymus. 

PUBLIC HEALTH RELEVANCE: The thymus is the primary organ where T lymphocytes are made, 

and thus is critical for normal immune function. Specialized cells inside the thymus (stromal cells) are 

responsible for instructing stem-like cells recruited from the bone marrow to undergo development into 

T lymphocytes. However, early in development, these stromal cells themselves require signals from 

immature lymphocytes to induce their own growth and development. In the absence of lymphocytes, for 

instance, as occurs in many pediatric immunodeficiency disorders, the thymus does not form. The 

goals of this project are to perform an in depth analysis of this very poorly understood two-way interplay 

between lymphocytes and stromal cells in the thymus, and how these processes shape the 

development of the immune system. 

CRITIQUE 1: 
 

 

 

 

 

 

 

1 R21 AI095831-01 3 CMIB 

PETRIE, H 

Significance: 1 

Investigator(s): 1 

Innovation: 1 

Approach: 2 

Environment: 3 

Overall Impact: This is a very strong proposal from a talented and experienced investigator who has 

provided key conceptual insights underlying the generally accepted paradigm that the thymus is a 

series of linked environments, and for the ordered sequence of migration events undertaken by T 

lymphocyte progenitors. The PI proposes to characterize the response of the IL7R deficient thymic 

stromal compartment to IL7R+ hematopoietic progenitor cells, using an elegant and exhaustive 

approach detailed in a recent publication in Immunity. In response to IL7R+ cells, it is known the 

thymus grows, with large numbers of proliferating stromal cells responding to the influx of competent 

progenitors. However, the signals delivered by progenitors to stromal cells are not well understood. PI 

proposes to isolate thymic stromal cells at different times after reconstitution by laser capture, subject 

them to microarray-based gene profiling, and subtract the signature simultaneously obtained of the 

lymphoid components of the thymus. The question is outstanding; the approach is state of the art and 

very likely to provide important insights. 

1. Significance: 

Strengths 

• Highly significant general question of how the thymic stromal compartment senses and 

responds to competent T cell progenitors. 

• Knowledge important for studies of thymic organogenesis, homeostasis, involution, and T cell 

positive and negative selection. 

Weaknesses 

2. Investigator(s): 

Strengths 

• Long history of interest in thymic development 

• Great expertise has consistently pushed this field forward. 

• Recent accomplishments include a key paper in Immunity that is expected to energize the field 

of thymic development, function, and thymic aging. 

3. Innovation: 

Strengths 

• The innovation of this proposal is the focus on signaling into thymic stromal cells during thymic 

growth, together with the recent development of key technical breakthroughs that make this 

study possible. 

4. Approach: 

Strengths 

• Builds on published work recently appearing in Immunity, in which gene expression of specific 

regions of the thymus are determined by first isolating these areas by laser capture, and 

microarraying them, and revealing the stroma-specific expression by subtracting out the 

lymphoid signature. The PI proposes to extend this to describe gene expression in cortical and 
 

 

 

1 R21 AI095831-01 4 CMIB 

PETRIE, H 

medullary IL7R-/- stromal cells when responding to an influx of competent progenitors. The 

stromal cells must sense and respond to IL7R+ progenitors because they increase cell division. 

In mice with profound blocks in T cell development, medullary epithelial compartments are 

almost entirely absent, however, some of the signals here are already know, as RANK: RANKL 

interactions between mature T cells and medullary TEC are known to drive expansion of mTEC. 

However, signals driving cortical growth are largely unknown. 

Weaknesses 

• The chief potential weakness of this proposal is that it is unclear whether the key signaling pairs 

that are important for signaling from lymphoid cells into stromal cells will be uncovered. Indeed, 

given that the IL7R-/- thymus can respond to incoming progenitors, the essential signaling 

molecules must be already expressed by IL7R-/- stromal cells. For these specific molecules to 

be captured by this exploratory approach, their expression will need to be further upregulated 

during thymic reconstitution, or unique signals downstream of these molecules that are 

upregulated will need to identify that allow the upstream triggers to be deduced. However, this is 

an acceptable level of risk for this potentially high reward R21 proposal. 

5. Environment: 

Strengths 

• Adequate for these studies. 

Weaknesses 

Protections for Human Subjects: Not Applicable (No Human Subjects) 

Vertebrate Animals: Acceptable 

• Adequate justification provided was included. 

Biohazards: Acceptable 

Resource Sharing Plans: Acceptable 

Budget and Period of Support: Recommend as Requested 

CRITIQUE 2: 

Significance: 1 

Investigator(s): 1 

Innovation: 2 

Approach: 1 

Environment: 3 

Overall Impact: This is a very strong application from a first rate investigator who proposes to 

characterize changes in gene expression in thymic stromal cells in a model of thymic organ growth, 

then, using sophisticated computer algorithms, to define possible important receptor-ligand pairs that 

could control thymic stroma-developing thymocytes cross-talk, and test a few of these resulting 

signaling axes in vivo. The investigator has previous experience with the biological and informatics 
 

 

 

 

 

 

 

1 R21 AI095831-01 5 CMIB 

PETRIE, H 

approaches, so feasibility is high. Given how little we know about thymic stroma, the results are very 

likely to have a big impact in the field. The investigator is uniquely qualified to perform this project. 

1. Significance: 

Strengths 

• The cross-talk between developing thymocytes and the stroma that supports them is known to 

be critical for development of the immune system, but its molecular basis are not well-defined. If 

successful this application would dramatically expand our knowledge in this area. 

Weaknesses 

2. Investigator(s): 

Strengths 

• Leader in the field. 

• Experience with these kind of approaches 

• Longstanding bioinformatics collaborator 

Weaknesses 

3. Innovation: 

Strengths 

• We know very little about how developing lymphoid cells influence stromal cell growth, 

differentiation and survival. The area of research is therefore innovative. 

• The research approach is relatively novel, although it does not break new ground in respect to 

the investigator’s previous publication. 

Weaknesses 

4. Approach: 

Strengths 

• Good experimental model. 

• Well designed strategy for the analysis and validation of the profiling results. 

Weaknesses 

5. Environment: 

Strengths 

• Strong bioinformatics community. 

Weaknesses 

• Small immunology community. 

• Little institutional support 

Vertebrate Animals: Acceptable 

• There are no concerns . 
 

 

 

 

 

 

 

 

 

 

1 R21 AI095831-01 6 CMIB 

PETRIE, H 

Resource Sharing Plans: 

• For an application likely to generate large amount of data of interest for the general community 

the resource sharing plan is too vague. A plan to make a database of the results accesible to 

other investigator would strenghthen this aspect of the application. 

Budget and Period of Support: Recommended budget 

• Appropriate 

CRITIQUE 3: 

Significance: 1 

Investigator(s): 1 

Innovation: 1 

Approach: 2 

Environment: 1 

Overall Impact: This R21 application from an experienced senior investigator seeks to identify the 

signals produced by developing T cells that induce the maturation of stromal elements in the thymus. 

These studies are highly significant as essentially nothing is known about how developing T cells signal 

stroma. Understanding how lymphoid cells induce thymic stromal responses has been a subject of 

contemplation for many laboratories for many years. However, given the difficulties associated with 

thymic stromal cell isolation, and the further limitation imposed by the small number of cells present in 

the immunodeficient thymus, it has been a very difficult issue to address. This application has 

established a novel, comprehensive, bioinformatics approach that will enable the identification of 

receptor-ligand pairs expressed by thymocytes and stroma without disrupting thymic structure, which 

has been shown to alter the expression of Notch ligands expressed by stroma. Therefore this proposal 

is innovative – both regarding focus and methods. The only caveat is that testing the function of stromal 

molecules can only be done by making conditional knockout mice which is costly and time-consuming. 

Nevertheless, this approach fits perfectly with the high-risk, high-reward mandate of the R21 

mechanism. 

THE FOLLOWING RESUME SECTIONS WERE PREPARED BY THE SCIENTIFIC REVIEW 

OFFICER TO SUMMARIZE THE OUTCOME OF DISCUSSIONS OF THE REVIEW COMMITTEE ON 

THE FOLLOWING ISSUES: 

VERTEBRATE ANIMAL (Resume): ACCEPTABLE 

MODEL ORGANISM SHARING PLAN: New model organisms will be generated during the project 

period and the application includes an adequate model organism sharing plan. 

COMMITTEE BUDGET RECOMMENDATIONS: The budget was recommended for time and 

amount as requested. 

NIH has modified its policy regarding the receipt of resubmissions (amended applications). 

See Guide Notice NOT-OD-10-080 at http://grants.nih.gov/grants/guide/notice-files/NOT-OD-
 

1 R21 AI095831-01 7 CMIB 

PETRIE, H 

10-080.html. 

The impact/priority score is calculated after discussion of an application by averaging the 

overall scores (1-9) given by all voting reviewers on the committee and multiplying by 10. The 

criterion scores are submitted prior to the meeting by the individual reviewers assigned to an 

application, and are not discussed specifically at the review meeting or calculated into the 

overall impact score. For details on the review process, see 

http://grants.nih.gov/grants/peer_review_process.htm#scoring.