Research Services presents

1. Research Servicespresents Study Section Trends:Re-submissions Emcee: Toni D’Agostino, Director, Office of Sponsored Programs

2. Agenda Welcome, Announcements and Introductions Panelists’ Presentations Discussion Study Section Trends

3. Study Section Trends • Moderator Miles Cloyd, Ph.D. • Professor, Microbiology & Immunology • Panelists Tracy Toliver-Kinsky, Ph.D. • Associate Professor, Anesthesiology Satish Srivastava, Ph.D. • Professor, Biochemistry & Molecular Biology

4. Grant Resubmissions: Approach to critiques and revisions Tracy Toliver-Kinsky, PhD Associate Professor Anesthesiology

5. Grant Resubmissions: Approach to critiques and revisions • Call your program director • Response to reviewers • Revisions

6. Grant Resubmissions: Approach to critiques and revisions Points to remember: • You have only two more chances to get the application funded • “Burden of proof” is on you • Things that may seem obvious to you may not be to the reviewer • Application may be read quickly and only once, so your writing must be unambiguous • You do not want to offend your reviewers, even if they are wrong

7. Grant Resubmissions: Approach to critiques and revisions Step #1: Call your program director

8. Response to reviewers • Brief introduction • thank the reviewers for their suggestions • brief summary of revisions (if possible) • indicate how changes are marked in application • Respond directly to every concern the reviewers raised • Tip: paste every concern, number them, then respond point-by-point

9. Response to reviewers • Brief introduction • thank the reviewers for their suggestions, • brief summary of revisions (if possible) • statement of how changes are marked in application Example: “Introduction to Revised Application We would like to thank the reviewers for their enthusiasm regarding this project, their thoughtful criticisms, and for the useful suggestions. Reviewer #1 was concerned with the design of the immunization schedule in aim 1, the time frame of cytokine assessments in aim 2, and the ability to successfully perform adoptive transfer of immune cells in aim 3. Reviewer #2 was predominantly concerned with the characterization of dendritic cells before and after treatment, and with potentially adverse/systemic side effects of Flt3L treatments. We have responded to all concerns in a point-by-point replyand haverevised the application accordingly. Changes to the original application are marked by a vertical line in the left margin.”

10. Response to reviewers Respond directly to every concern the reviewer raised: describe revisions Tip: paste every concern, number them, then respond point-by-point • Reviewer 1 • “There is some concern about the way these immunization studies are designed. The mice will be immunized 1 week prior to injury, then boosted with TT at 13 days after injury. This approach will indicate how injury influences an ongoing T-cell response rather than a response that will be influenced by the injury or FLT3L treatment.” We would like to thank the reviewer for recognizing the flaw in the previous immunization schedule. We have redesigned this experiment with two different immunization protocols that will permit dissection of the effects of injury and Flt3L treatments on both the primary and secondary (recall) humoral responses to antigen (see Figure 15). The immunization schedules are described in detail in experiment 1-1 and in Figure 15, and we include preliminary data showing that a 4 week rest period is sufficient to complete the primary IgG and IgM response to tetanus toxoid (Figure 14). • 2) “Also, there is no mention of what adjuvant will be used.” The tetanus toxoid preparation to be used for immunizations will be alum-adsorbed (aluminum hydroxide). This is a commonly used adjuvant for tetanus toxoid immunizations1. Details have been incorporated into the Methods section.

11. Response to reviewers • Valid concerns: agree, address and fix, and locate changes for the reviewer 5) “The strength of the studies in this specific aim [2] is that they will be phenotyping innate and adaptive immune response changes in response to infection. Their proposed approaches include RNAse protection assays and serum cytokine bead array studies…However, it is not clear at what time points they will be making their determinations.”We regret the lack of detail originally provided and have incorporated this information. We will examine cytokine responses in tissues and sera 2, 3, and 4 days after wound inoculation, which will permit an examination of cytokine responses 24 hours prior to and during the time frame of mortality due to burn wound infection. The rationale for these time points is provided in the Research Design and Methods section under experiment 2-2.

12. Response to reviewers Invalid concerns: delicately disagree and thoroughly defend *remember: maybe you didn’t explain it clearly enough! 6) “The last specific aim will address which cell populations are involved in the beneficial effects of FLT3L treatment…. If successful, this approach will indicate whether DCs alone are responsible for the improved immunity following FLT3L treatment or whether it is due to increased NK cell function. However, there are many adoptive transfer studies proposed with no supporting information to indicate that these studies are feasible….Since these studies will depend mostly on successful adoptive transfer, more supporting information is needed to verify the feasibility of these proposed studies.” We have included new datashowing the presence of CFSE-labeled dendritic and NK cells in the spleen (Figure 10) 48 hours after i.p. injection. In addition to demonstrating the presence of transferred cells after i.p. injection, we demonstrate a functional effect after i.p. transfer of dendritic cells (protection from lethal wound infection,figure 10)and have previously demonstrated a functional effect after i.p. transfer of NK cells (restoration of mortality after rapidly lethal CLP in β2-microglobulin mice treated with anti-asialoGM1)2. Although intravenous transfer of cells is common, the use of i.p. injection for adoptive transfer is not uncommon and has been reported for transfer of numerous leukocytes including macrophages3, dendritic cells4, T cells5,6, and total splenocytes7,8. One group performed a direct comparison of i.p. and intravenous administration of B cells and found no differences in the trafficking patterns or level of tissue reconstitution9. Our preliminary data indicate that enough dendritic and NK cells can be transferred by intraperitoneal injection to be visualized in the spleen and, most importantly, to induce a functional effect.

13. Response to reviewers • Invalid concerns: delicately disagree and thoroughly defend • *Remember: maybe you didn’t explain it clearly enough! 6) “One concerning aspect about this aim is the animal studies proposed in aim 3.5. All of their work has been completed thus far in BALB/C mice. They state that the transgenic mice have been bred onto the BALB/C background but it is important that the controls for these studies are appropriate for the experimental group.” The CD11c-DTR transgenic mice were bred on a BALB/c background so we anticipate similar responses to Flt3L, burn, and wound infection in the CD11c-DTR mice as in the BALB/c mice that were used in our prior studies. Nonetheless, as stated in the methods, control groups will consist of CD11c-DTR transgenic mice that will be treated with Flt3L but not injected with diphtheria toxin, and CD11c-DTR mice that receive no Flt3L and no diphtheria toxin prior to wound infection. We have expanded the description of these control groups in the research design section (experiment 3-5).

14. Revisions • Clearly mark revisions in the application (underline, italics, margin lines) • Be especially thorough, since the reviewers will focus most of their time on revisions versus entire application • Remember, this is your only communication with the reviewers, and you only have two more chances to submit!

15. $ $ $ $ Good luck! $ $ $ $ $ $

16. Grant Resubmissions: Senior scientist and study section perspective Satish Srivastava, PhD Professor Biochemistry & Molecular Biology

17. Grant Resubmissions: Overview & Summary Miles Cloyd, PhD Professor Microbiology & Immunology

18. Research Services Thank you. Questions from the audience.