PI: MCCUNE, JOSEPH M Title: Human immune system layering and the neonatal response to vaccines

Received: 06/14/2010 FOA: PA10-069 Council: 01/2011

Competition ID: ADOBE-FORMS-B FOA Title: NIH EXPLORATORY DEVELOPMENTAL RESEARCH GRANT 

PROGRAM (PARENT R21)

1 R21 AI094009-01 Dual: Accession Number: 3306088

IPF: 577508 Organization: UNIVERSITY OF CALIFORNIA SAN FRANCISCO

Former Number: Department: Medicine

IRG/SRG: IHD AIDS: N Expedited: N

Subtotal Direct Costs 

(excludes consortium F&A) 

Year 1:

Year 2:

Animals: N 

Humans: Y 

Clinical Trial: N

Current HS Code: 30

HESC: N

New Investigator: N

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Senior/Key Personnel: Organization: Role Category:

Joseph McCune The Regents of the University of 

California, San Francisco

PD/PI 

Shannon Thyne MD The Regents of the University of 

California, San Francisco

Co-Investigator

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The Regents of the University of California, San Francisco

3333 California Street

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Giovannetti

Human immune system layering and the neonatal response to vaccines

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McCune

Joseph M.

941103518

San Francisco

Bldg. 3, Rm 601

Experimental Medicine

San Francisco

1001 Potrero Avenue

Medicine

Professor of Medicine

The Regents of the University of California, San Francisco

CA: California

USA: UNITED STATES

	


 		

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424 R&R and PHS-398 Specific

Table Of Contents Page Numbers

SF 424 R&R Face Page------------------------------------------------------------------------------------------ 1

Table of Contents--------------------------------------------------------------------------------------------- 3

Performance Sites--------------------------------------------------------------------------------------------- 4

Research & Related Other Project Information------------------------------------------------------------------ 5

 Project Summary/Abstract (Description)---------------------------------------- 6

 Public Health Relevance Statement (Narrative attachment)---------------------------------------- 7

 Facilities & Other Resources---------------------------------------- 8

 Equipment---------------------------------------- 9

Research & Related Senior/Key Person-------------------------------------------------------------------------- 10

 Biographical Sketches for each listed Senior/Key Person---------------------------------------- 11

PHS 398 Specific Cover Page Supplement------------------------------------------------------------------------ 19

PHS 398 Specific Modular Budget------------------------------------------------------------------------------- 21

 Personnel Justification---------------------------------------- 24

 Additional Narrative Justification---------------------------------------- 25

PHS 398 Specific Research Plan-------------------------------------------------------------------------------- 26

 Specific Aims---------------------------------------- 27

 Research Strategy---------------------------------------- 28

 Human Subjects Sections---------------------------------------- 34

 Protection of Human Subjects---------------------------------------- 34

 Women &Minorities---------------------------------------- 36

 Planned Enrollment Table---------------------------------------- 37

 Children---------------------------------------- 38

 Bibliography & References Cited---------------------------------------- 39

 Letters of Support---------------------------------------- 41

 Resource Sharing Plan---------------------------------------- 46

PHS 398 Checklist--------------------------------------------------------------------------------------------- 47

Table of Contents Page 3

Principal Investigator/Program Director (Last, first, middle): McCune, Joseph, M.
County:

* ZIP / Postal Code:

* Country:

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Street2:

Organization Name:

Project/Performance Site Location(s)

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University of California, San Francisco

1001 Potrero Avenue

Bldg. 3, Room 627

San Francisco San Francisco

CA: California

USA: UNITED STATES

941103518

OMB Number: 4040-0010 

Expiration Date: 08/31/2011

I am submitting an application as an individual, and not on behalf of a company, state, 

local or tribal government, academia, or other type of organization. 

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Performance Sites Page 4

Principal Investigator/Program Director (Last, first, middle): McCune, Joseph, M.
1. * Are Human Subjects Involved?

IRB Approval Date:

Human Subject Assurance Number:

2. * Are Vertebrate Animals Used?

IACUC Approval Date:

Animal Welfare Assurance Number

4.b. If yes, please explain:

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 environmental impact statement (EIS) been performed?

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6.b. Optional Explanation:

7. * Project Summary/Abstract

11. Equipment

8. * Project Narrative

12. Other Attachments

RESEARCH & RELATED Other Project Information

Is the IACUC review Pending?

If no, is the IRB review Pending?

2.a. If YES to Vertebrate Animals

3. * Is proprietary/privileged information included in the application? 

4.a. * Does this project have an actual or potential impact on the environment? 

6.a. If yes, identify countries:

1 2 3 4 5 6

9. Bibliography & References Cited

10. Facilities & Other Resources

Yes No

1.a If YES to Human Subjects

Yes No

Yes No

If yes, check appropriate exemption number.

Yes No

Yes No

Yes No

Yes No

Yes No

Yes No

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Is the Project Exempt from Federal regulations?

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5.a. If yes, please explain:

 


  		

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Other Information Page 5

Principal Investigator/Program Director (Last, first, middle): McCune, Joseph, M.
PROJECT SUMMARY

The development of the mammalian immune system is typically thought to occur in a linear fashion, from 

immaturity to maturity as a function of antigen exposure. Previous findings in birds and in mice, however, 

indicate that this view is oversimplified. Thus, in these species, the developing immune system appears to be

“layered” in a manner that is independent of antigen exposure, beginning as a multilineage fetal system that 

is replaced by an anatomically and biologically distinct multilineage system after birth. If so, then

developmentally ordered and unique hematopoietic stem/progenitor cells (HSPC) could give rise to distinct 

lymphocyte lineages at different stages of development. 

In ongoing experiments, we have found that such immune system “layering” occurs in humans. Our 

preliminary data show that a vigorous human fetal immune response to exogenous antigens can be actively 

suppressed by antigen-specific Tregs, that these fetal Tregs are derived from a fetal-specific lineage of T 

cells, and that this lineage is generated by an HSPC that is distinct from that found in adults. These data 

suggest that the human immune system is comprised of two distinct waves: one generated from a “fetal” 

HSPC that exists in utero in the fetal liver and bone marrow, and another generated from a superseding 

“adult” HSPC that resides in the bone marrow at later time points. The former gives rise to an immune 

system that is prone to deliver a tolerogenic response to foreign antigens. The latter gives rise to an immune 

system that is more likely to generate an immunoreactive responses (e.g., one including cytotoxic T cells and 

neutralizing antibodies). 

Given these findings, we hypothesize that physiologic layering of immune system ontogeny leads to a normal 

range in the ratio of fetal- to adult-type T cells at birth, with some neonates exhibiting a higher fraction of fetal 

T cells than others; and that those with a high ratio of fetal/adult T cells will generate predominant Th2 

responses to routine childhood immunizations. These hypotheses will be addressed in the experiments of the 

following Specific Aims: (1) to determine the normal range of fetal to adult T cells in the umbilical cord blood 

of the full term neonate; and (2) to determine whether those full term neonates with a high ratio of fetal/adult 

T cells are more likely to generate a Th2-polarized immune response to routine childhood vaccines.

Should this exploratory study reveal normal variation in the ratio of fetal to adult T cells at birth and should 

such variation be directly related to a Th2 skew after childhood vaccination, modalities aimed at changing this 

ratio more towards the adult lineage at birth may provide benefit to a substantial number of newborns.

Project Description Page 6

Principal Investigator/Program Director (Last, first, middle): McCune, Joseph, M.
PROJECT NARRATIVE

These exploratory studies are relevant to public health for two reasons. First, they may provide proof-ofconcept evidence for the existence of a range in the extent of immune system “layering” in human neonates. 

Secondly, they may demonstrate that this range is itself related to (and possibly causal of) differences in the 

ability of neonates to withstand infections and to respond to vaccines. 

Public Health Relevance Statement Page 7

Principal Investigator/Program Director (Last, first, middle): McCune, Joseph, M.
FACILITIES & OTHER RESOURCES

Laboratory: The Division of Experimental Medicine (DEM) is located within laboratories built in 1991 on the 

San Francisco General Hospital (SFGH) campus of University of California at San Francisco (UCSF). This 

facility occupies 27,000 square feet of laboratory space arranged in multiple bays to promote scientific 

interaction and includes several specialized multi-user facilities: a customized 4-laser, air-launched, 19-

parameter BD FACSAria cell sorter for the phenotypic analysis and purification of various HIV-infected and 

uninfected cells; two customized, 4-laser BD LSRII flow analyzers set up to read 14 or 19 parameters; a 

phosphorimager to facilitate rapid quantitative analysis of autoradiographic samples; confocal and electron 

immunofluorescent microscopes for cell analysis. Additional facilities within the Division include a 1,500 

square foot biosafety level 2 (BSL2*) containment laboratory for the safe handling of HIV and other infectious 

agents, standard (BSL2) tissue culture facilities on each of the two research floors, and three large cold 

rooms. The facility also has a conference room/library with seating for more than 70 people and fully 

equipped for video relay of major seminars from the other UCSF campuses. Dr. McCune has a fully 

equipped laboratory with 15 benches/desks.

Clinical: The DEM is located next door to SFGH, a public hospital and regional trauma center fully equipped 

with modern diagnostic instruments, a clinical laboratory which processes routine as well as nonstandard 

laboratories on a round-the-clock basis, and ancillary services capable of handling most medical conditions 

on an out- and/or in-patient basis. The clinical resources of SFGH include the Clinical and Translational 

Science Institutes (CTSI) Clinical Research Center (CCRC) with 10 inpatient beds and an outpatient facility. 

Ancillary support for a variety of other important clinical and translational research needs have been 

established by the UCSF CTSI, including recharge services for statistical consultation for study design, data 

analysis, and database management; assistance in regulatory requirements for human subjects; and core 

laboratories offering sample processing and aliquoting, immunologic assays, virologic assays, DNA 

extraction, restriction fragment length polymorphism analysis, and special instruments, devices or services 

(including high performance liquid chromatography, dual energy x-ray absorptiometry, treadmill and exercise 

physiology, and indirect calorimetry). 

Animal: Not applicable.

Computer: A completely networked computer system with hardware connections to the greater UCSF 

system is available to all personnel for scientific writing, editing, data nucleic acid and protein data analysis, 

and graphics illustration. It is equipped with a complete set of sequence analysis programs, including 

GeneWorks, DNA Strider, access to sequence databases, and an in-house reference library. This allows the 

DEM staff to access mainframe computers dedicated to sequence analysis and database storage in the 

international Internet network, which provides E-mail and file transfer capabilities as well. Access to the 

MEDLINE literature database is also available to every computer in the DEM via this Internet connection. Dr. 

McCune has a desktop computer networked to the University server.

Office: Dr. McCune has a furnished office in close proximity to his laboratory, In addition, the Division is 

staffed by four full time administrators.

Other: The DEM was created (as of 3/1/06) by the Department of Medicine at UCSF, with its laboratory 

space based at San Francisco General Hospital, not far from the new Mission Bay campus. The Division 

Chief is Dr. Joseph M. McCune and the Associate Chief is Dr. Doug Nixon. Other faculty members are Drs. 

Cheryl Stoddart, Elizabeth Sinclair, Mary Premenko-Lanier, and Maggie Feeney. The Division is currently 

accepting applications from physician scientists and PhD scientists engaged in hypothesis-driven, patient- or 

disease-oriented research on the immunology of chronic infectious diseases in humans. Over the course of 

the next several years, it is anticipated that 2-3 more appointments will be made at the Assistant to Full 

Professor level in the In Residence series, depending upon qualifications. Successful candidates will also 

become members of the UCSF Graduate Program in Biomedical Sciences. All the support services of a 

major University campus are available to this project: library, computer center, material management, 

accounting, business, and fiscal and personnel services.

Facilities Page 8

Principal Investigator/Program Director (Last, first, middle): McCune, Joseph, M.
EQUIPMENT

Major equipment available in the Division of Experimental Medicine includes: a customized 4-laser, airlaunched, 19-parameter BD FACSAria cell sorter for the phenotypic analysis and purification of various HIVinfected and uninfected cells; two customized, 4-laser BD LSRII flow analyzers set up to read 14 or 19 

parameters; a phosphorimager to facilitate rapid quantitative analysis of autoradiographic samples; confocal 

and electron immunofluorescent microscopes for cell analysis; DNA sequencing equipment (including an 

Applied Biosystems 373A fluorescence-based DNA sequencing system); ultracentrifuges; low- and high-speed 

centrifuges, immunofluorescence microscope with digital camera; autoclaves; X-ray processor; bacterial 

incubators/shakers; liquid scintillation counter; FPLC; spectrophotometer; thermal cyclers; and miscellaneous 

small equipment. The equipment is located either directly in the laboratory or in adjacent equipment rooms.

Equipment Page 9

Principal Investigator/Program Director (Last, first, middle): McCune, Joseph, M.
Province:

PROFILE - Project Director/Principal Investigator

Prefix: * First Name: Middle Name:

* Last Name: Suffix:

Organization Name: Division:

Position/Title: Department:

* Street1:

Street2:

* Phone Number: Fax Number:

* E-Mail:

Credential, e.g., agency login:

* Project Role: Other Project Role Category:

* Country: * Zip / Postal Code:

* State:

* City: County/ Parish:

Attach Current & Pending Support

RESEARCH & RELATED Senior/Key Person Profile (Expanded)

*Attach Biographical Sketch

Joseph M.

McCune

Professor of Medicine Medicine

The Regents of the University of California, San Francisco

1001 Potrero Avenue

San Francisco San Francisco

Experimental Medicine

Bldg. 3, Rm 601

CA: California

USA: UNITED STATES 941103518

PD/PI

McCuneSF424Bio1011440819.pdf Add Attachment Delete Attachment View Attachment

Add Attachment Delete Attachment View Attachment

OMB Number: 4040-0001 

Expiration Date: 06/30/2011

Degree Type:

Degree Year:

Province:

PROFILE - Senior/Key Person

Prefix: * First Name: Middle Name:

* Last Name: Suffix:

Organization Name: Division:

Position/Title: Department:

* Street1:

Street2:

* Phone Number: Fax Number:

* E-Mail:

Credential, e.g., agency login:

* Country: * Zip / Postal Code:

* State:

* City: County/ Parish:

MD

M.

Assistant Clinical Professor

The Regents of the University of California, San Francisco

San Francisco

1001 Potrero Ave, Mail Stop 6D

General Pediatrics

Pediatrics

USA: UNITED STATES

Shannon

Thyne

SFGH, Dept. of Pediatrics

San Francisco

CA: California

941103518

* Project Role: Co-Investigator Other Project Role Category:

1

Degree Type:

Degree Year:

*Attach Biographical Sketch ThyneSF424Bio1011440775.pdf Add Attachment Delete Attachment View Attachment

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Key Personnel Page 10

Principal Investigator/Program Director (Last, first, middle): McCune, Joseph, M.
BIOGRAPHICAL SKETCH

Provide the following information for the key personnel and other significant contributors.

Follow this format for each person. DO NOT EXCEED FOUR PAGES.

NAME

McCune, Joseph M.

POSITION TITLE

Professor of Medicine, University of California, San 

Francisco

Chief, Division of Experimental Medicine eRA COMMONS USER NAME

EDUCATION/TRAINING (Begin with baccalaureate or other initial professional education, such as nursing, and include postdoctoral training.)

INSTITUTION AND LOCATION DEGREE

(if applicable) YEAR(s) FIELD OF STUDY

Harvard College A.B. 1975 Biochemistry

The Rockefeller University Ph.D. 1981 Cell Biology and 

Immunology

Cornell Medical College M.D. 1982 Medicine

A. Personal Statement

The goal of this exploratory study is to determine whether the human immune system shows “layering” during 

ontogeny, with a tolerogenic fetal system that is superseded by an “adult-type” system that is more likely to 

respond against foreign antigens. If so, then we hypothesize that those neonates born with a larger component 

of the “fetal-type” system at birth will be less capable of responding to childhood immunizations with a strong 

Th1-type response and more likely to succumb to infections and/or atopic disorders. This hypothesis will be 

studied in the context of a cross-sectional analysis of 150 cord blood specimens and a prospective study of 50 

cord blood specimens. These are studies that are consistent with the thrust of my research over the past 20 

years and that will take advantage of existing collaborations, approved human subjects protocols, and 

standardized techniques. They will be conducted within the Division of Experimental Medicine, a new division 

in the UCSF Department of Medicine designed to foster a multidisciplinary environment for the pursuit of 

hypothesis-driven studies in human subjects. 

B. Positions and Honors

Positions: Resident in Internal Medicine, University of California, San Francisco (UCSF) (1982-84); Infectious 

Disease Fellow, UCSF (1984-86); Postdoctoral Fellow, Department of Pathology, Stanford University School of 

Medicine (1985-88); Physician, UCSF AIDS Clinic, San Francisco General Hospital (SFGH) (1986-present); 

Scientific Director, SyStemix, Inc. (1988-91); Vice-President, New Enterprise Research Division, SyStemix, Inc. 

(1991-94); Director: SyStemix-Sandoz Antiviral Project & SyStemix-Sandoz HIV Gene Therapy Joint Venture 

(Progenesys, Inc) (1993-94); Associate Investigator, Gladstone Institute of Virology and Immunology (GIVI) 

(1995-1999); Senior Investigator, GIVI (1999-2006); Associate Professor of Medicine, UCSF (1995-1999); 

Associate Director, General Clinical Research Center, SFGH (1995-2006); Visiting Professor (on sabbatical), 

Institut Pasteur, Paris, France (2003-2004); Director and PI of the UCSF Clinical and Translational Science 

Institute (2006-2008); Senior Associate Dean of Clinical and Translational Research (2005-2008); Chief of the 

Division of Experimental Medicine (2006-present); Professor of Medicine, UCSF (1999-present)

Member: Project Inform Immune Restoration Think Tank (1992-present); Health Advisory Board, Pediatric 

AIDS Foundation (1994-present); National Board of Governors, Project Inform (1995-present); Councilor, The 

Henry Kunkel Society (1997-present); The Rockefeller University Board of Trustees (1998-2001); NIAID Study 

Section AIP (2000-present); UCSF Promotions Subcommittee (2000-2004); Chair, Scientific Advisory Board, 

Foundation of AIDS and Immunologic Research (2000-present); Chair, Gladstone Committee on Management 

and Mentoring Training (2001-present); Chair, UCSF Search Committee for Chair of GI, SFGH (2003); Chair, 

Translational Research Search Committee (2004-present); Elizabeth Glaser Pediatric AIDS Foundation Board 

of Directors (2006-present); Immune Tolerance Institute Board of Directors (2006-present)

Honors: National Merit Scholarship (1972-75); Harvard National Scholarship (1972-75); Michael C. Rockefeller 

Memorial Fellowship (1975); Pfizer Postdoctoral Fellowship (1986-89); Diplomate, Internal Medicine (1987); 

Elizabeth Glaser Scientist Award (1996-2000); Member, American Society for Clinical Investigation (1999); 

Member, American Association of Physicians (2000); Burroughs Wellcome Fund Clinical Scientist Award in 

Translational Research (2000-2005); NIH Merit Award (2001); NIH Director’s Pioneer Award (2004); NIH 

Biosketches Page 11

Principal Investigator/Program Director (Last, first, middle): McCune, Joseph, M.
Clinical and Translational Science Institute Award (2006); Pediatric Fellows and Fellows Leadership and 

Advocacy Group Mentorship Award, nominee (2007); UCSF Postdoctoral Scholar’s Association Outstanding 

Mentor Award (2007)

C. Selected Peer-reviewed Publications (from a total of 168)

1. Hellerstein M, Hanley MB, Cesar D, Siler S, Papageorgopoulos C, Wieder E, Schmidt D, Hoh R, Neese R, 

Macallan D, Deeks S, McCune JM. Directly measured kinetics of circulating T lymphocytes in normal and 

HIV-1-infected humans. Nature Med. 5:83-89, 1999.

2. McCune JM, Hanley MB, Cesar D, Halvorsen R, Hoh R, Schmidt D, Wieder E, Deeks S, Siler S, Neese R, 

Hellerstein M. Factors influencing T-cell turnover in HIV-1-seropositive patients. J. Clin. Invest. 105:R1-

R8, 2000. 

3. Huang SS, Barbour JD, Deeks SD, Huang JS, Grant RM, Ng VL, McCune JM. Reversal of Human 

Immunodeficiency Virus type 1-associated hematosuppression by effective antiretroviral therapy. Clin. 

Infect. Dis. 30:504-10, 2000.

4. Napolitano LA, Grant RM, Deeks SG, Schmidt D, De Rosa SC, Herzenberg LA, Herndier BG, Andersson J, 

McCune JM. Increased production of IL-7 accompanies HIV-1-mediated T-cell depletion: implications for 

T-cell homeostasis. Nature Med. 7:73-79, 2001.

5. Napolitano LA, Lo JC, Gotway MB, Mulligan K, Barbour JD, Schmidt D, Grant RM, Halvorsen RA, 

Schambelan M, McCune JM. Increased thymic mass and circulating naive CD4+ T cells in HIV-1-

infected adults treated with growth hormone. AIDS. 16: 1103-1111, 2002.

6. Hellerstein MK, Hoh RA, Hanley MB, Cesar D, Lee D, Neese RA, McCune JM. Subpopulations of long-lived 

and short-lived T-cells in advanced HIV-1 infection. J. Clin. Invest. 112: 956-966, 2003.

7. Napolitano LA, Burt T, Bacchetti P, Barron Y, French A, Kovacs A, Anastos K, Young M, McCune JM, 

Greenblatt RM. Increased circulating interleukin-7 levels in HIV-1-infected women. J. Acquir. Immune 

Defic. Syndr. 40:581-84, 2005.

8. Hartigan-O’Connor D, Abel K, McCune JM. Suppression of SIV-specific CD4+ T cells by infant but not adult 

macaque regulatory T cells: implications for SIV disease progression, J. Exp. Med., 204:2679-92, 2007.

9. Mold JE, Michaelsson J, Burt TD, Muench MO, Beckerman KP, Busch MP, Lee T-H, Nixon DF, and McCune 

JM. Maternal alloantigens promote the development of tolerogenic fetal regulatory T cells in utero. 

Science, 322:1562-65, 2008. PMCID: PMC2648820

10. Napolitano LA, Schmidt D, Gotway MB, Ameli N, Filbert EL, Ng MM, Clor JL, Epling L, Sinclair E, Baum 

PD, Li K, Killian ML, Bacchetti P, McCune JM. Growth hormone enhances thymic function in HIV-infected 

adults. J. Clin. Invest. 118:1085-98, 2008. 

. 

PMCID: PMC2248326

11. Favre D, Lederer S,* Kanwar B,* Min Ma Z,* Proll S, Kasakow Z, Mold J, Swainson L, Barbour JD, Baskin 

CR, Palermo R, Pandrea I, Miller C, Katze M, McCune JM. Critical loss of the balance between TH17 and 

T regulatory cell populations in pathogenic SIV infection. PLoS Pathogens, 5(2):e1000295, 2009 (*coequal authors). 

. 

PMCID: PMC2635016

12. Loke P,* Favre D,* Hunt PW, Leung JM, Kanwar B, Martin JN, Deeks SG, McCune JM. Correlating cellular 

and molecular signatures of mucosal immunity that distinguish HIV controllers from non-controllers. 

Blood, Feb 16, 2010. 

. 

13. L. Swainson, J. Mold, U. Bajpai, and M. McCune. 2010. Expression of the autoimmune susceptibility 

gene FcRL3 on human regulatory T cells is associated with dysfunction and high levels of 

programmed cell death-1. J Immunol 184: 3639-47. NIHMS # 207784.

PMCID: PMC2858476 [Available on 2011/4/15]

14. Stoddart CA, Keir ME, McCune JM. IFN-a-mediated induction of CCR5 leads to expanded HIV-1 tropism 

in vivo. PLoS Pathogens, 6:e1000766, 2010. PMCID: PMC2824759

15. Favre D,* Mold J,* Hunt P, Kanwar B, Loke P, Seu L, Barbour J, Lowe MM, Jayawardene A, Aweeka F, 

Huang Y, Douek D, Brenchley JM, Martin JN, Hecht FM, Deeks SG, McCune JM. Tryptophan catabolism 

by indoleamine 2,3-dioxygenase 1 alters the balance of Th17 and regulatory T cells in HIV disease. 

Science Translational Medicine, 2:32ra36, 2010. (*co-equal authors). 

. 

D. Research Support

Current Research Support

DP1 OD000329 (PI: McCune) 09/30/04–07/31/10 

NIH Director’s Pioneer Award

This project will address the need to understand patterns of an effective immune response to HIV in humans, 

with the intent to inform the creation of an effective vaccine. 

Biosketches Page 12

Principal Investigator/Program Director (Last, first, middle): McCune, Joseph, M.
R37 AI40312 (PI: McCune) 04/01/96–02/28/11 

NIH/NIAID: HIV Effects on the Central Hematolymphoid System in vivo

The major goal of this project is to test the hypothesis that HIV infection leads to destruction and/or dysfunction 

of the bone marrow and thymic microenvironments which enable multilineage and lineage-committed 

hematopoiesis. 

 

P01 DA026134 (PI: Smith) 09/30/08-05/31/11

NIH/NIDA: HIV Proteomic Center for Host-Viral Response Characterization

The overall goals of this grant are to apply proteomics technologies to investigate changes in protein 

abundances resulting from the pathologic events associated with concomitant lentiviral infection and substance 

abuse as well as from pharmacologic interventions designed to treat these pathologies.

KL2 RR024130 (PI: Johnston) 09/30/06-06/30/11

UL1 RR024131

TL1 RR024129 

NIH: Clinical and Translational Science Institute

The purpose of this grant is to forge a uniquely transformative, novel, and integrative academic home for 

Clinical and Translational Science (CTSI). Dr. McCune was PI of the grant until a successor was named on 

12/22/08.

R01AI084109 (PI: McCune

NIH/NIAID: Interruption of Maternal-Fetal Transmission of HIV

) 09/05/09-08/31/13

The specific aim of this study is to demonstrate that the in utero exposure of the fetal rhesus macaque to 

SIV epitopes will induce a tolerogenic immune response that will protect against pathogenic SIV infection 

after birth.

Completed Research Support

U01 AI43864 (PI: McCune) 04/31/03-03/31/10

NIH/NIAID 

Thymic function in adults with HIV-1 disease

R01 AI47062 (PI: McCune) 07/01/03-12/31/08

NIH/NIAID 

Assessing laboratory correlates of immune protection

R21AI068583 (PI: McCune) 07/01/06-06/30/09 

NIH/NIAID 

Gag/anti-Gag immune complexes and enhancement of lentiviral disease progression

 

 

AI05418 (PI: Stoddart) 08/01/00-07/31/07

NIH/NIAID

Biosketches Page 13

Principal Investigator/Program Director (Last, first, middle): McCune, Joseph, M.
SCID-hu animal model for evaluating HIV therapies

McCune role on the project: Consultant.

Biosketches Page 14

Principal Investigator/Program Director (Last, first, middle): McCune, Joseph, M.
BIOGRAPHICAL SKETCH

Provide the following information for the key personnel and other significant contributors.

Follow this format for each person. DO NOT EXCEED FOUR PAGES.

NAME

Thyne, Shannon M.

POSITION TITLE

Associate Professor of Clinical Pediatrics 

eRA COMMONS USER NAME

EDUCATION/TRAINING (Begin with baccalaureate or other initial professional education, such as nursing, and include postdoctoral training.)

INSTITUTION AND LOCATION DEGREE

(if applicable) YEAR(s) FIELD OF STUDY

Yale College, New Haven, CT BA 05/89 Psychology

Brown University School of Medicine, 

Providence, RI (Brown-Dartmouth Program in 

Medicine)

MD 05/95 Medicine

A. Personal Statement

I am the Medical Director of a busy, urban, inner-city pediatric clinic based at San Francisco General Hospital, 

within the UCSF academic hospital network. This unit houses primary care, urgent care, and multiple subspecialty clinics providing care to a diverse and underserved patient population. This clinical setting has given 

me the opportunity to engage in clinical investigations in the areas of asthma, infectious diseases, adolescent 

health, child development, and improved systems of care. Over the past 10 years, I have collaborated with 

several well-established PIs on clinical research trials in these areas and have developed my own independent 

research projects in the area of pediatric asthma. Due to my ongoing connection to the clinical setting, I remain 

particularly adept at the design and implementation of feasible research projects, and am able to maintain 

consistent and close oversight of the research teams. The studies proposed by Dr. McCune in his R21 are 

exactly in line with the type of work that I’ve been able to carry out in my clinic in the past. I have no doubt but 

that I will be able to organize this effort going forward, should it be funded.

B. Positions and Honors

1995-1998 Resident, UCSF Department of Pediatrics 

Positions

1998-1999 Chief Resident, UCSF Department of Pediatrics

1999-2000 Clinical Instructor, Pediatrics, University of California, San Francisco

2000-2006 Health Sciences Assistant Professor, UCSF Department of Pediatrics

2001-present Medical Director, Child Protection Services, City of San Francisco

2001-present Medical Director, Children’s Health Center, San Francisco General Hospital

2006-2009 Health Sciences Associate Professor, UCSF Department of Pediatrics

2009-present Associate Professor of Clinical Pediatrics, UCSF Department of Pediatrics

1995 American Medical Women’s Association, Janet A. Glasgow Memorial Achievement

Selected Honors and Awards

 Citation 

1995 New England Pediatric Society, Pediatric Prize 

1996 Julius Krevans Award for Clinical Excellence in Pediatrics, San Francisco General Hospital

1997 Resident Teaching Award, UCSF Department of Pediatrics

1998 Moses Grossman Award for Community Service, UCSF Department of Pediatrics

2001 Hellman Family Award for Early Career Faculty, UCSF School of Medicine 

2002 Nominee, Kaiser Award for Excellence in Teaching, UCSF School of Medicine

2003 Nominee, Medical Student Award for Excellence in Small Group Instruction, UCSF School of 

Medicine

2004 UCSF Medical Student Award for Excellence in Small Group Instruction

2005 AMA Hospital Recognition Program, “Innovative Approaches to Patient-Centered Communication” 

for Pediatric Asthma Clinic at San Francisco General Hospital

Biographical Sketches for each listed Senior/Key Person 2 Page 15

Principal Investigator/Program Director (Last, first, middle): McCune, Joseph, M.
2006 UCSF Academy of Medical Educators Award for Excellence in Innovative Curricular Design and 

Program Development

2006 San Francisco Department of Public Health, Award of Excellence, for Best Practices in Cultural 

Competency Training, Advancing Cultural Competency in the San Francisco Department of Public 

Health

2007 Ambulatory Pediatric Association, Health Care Delivery Award (The Yes We Can Urban Asthma 

Partnership at UCSF/San Francisco General Hospital)

2008 San Francisco Health Plan, Award for Clinical Excellence in Pediatric Asthma Care

2009 San Francisco Health Plan, Overall Award for Excellence in Pediatric Primary Care (Fluoride 

Varnish Program)

C. Selected Peer-reviewed Publications

1. Tsai, HJ, Kho, JY, Shaikh N, Choudry S, Naqvi M, Navarro D, Matallana H, Castro R, Lilly CM, Watson 

HG, Meade K, Lenoir M, Thyne S, Ziv E, Burchard EG; Admixture-matched case-control: a practical 

approach for genetic association studies in admixed populations. Human Genetics. 2006 January; 

118(5):626-39.

2. Tsai, HJ, Shaikh, N, Kho, JY, Battle N, Naqvi M, Navarro D, Matallana H, Lilly CM, Eng CS, Kumar G, 

Thyne S, Watson HG, Meade K, Lenoir M, Choudhry S, Burchard EG; from the Study of African 

Americans, Asthma, Genes Environments (SAGE); Beta(2)-Adrenergic receptor polymorphisms: 

pharmacogenetic response to bronchodilator among African American asthmatics. Human Genetics. 2006 

June; 119(5):547-57.

3. Thyne SM, Rising J, Legion V and Love MB. The Yes We Can Urban Asthma Partnership: an innovative 

approach to childhood asthma management. Journal of Asthma, 2006 October, 43:667-673. 

4. Cabana, MD and Thyne SM. An emergency department-based follow-up clinic can improve asthma 

outcomes [Invited Commentary]. Journal of Pediatrics, 2006 November, 149 (5), 725.

5. Thyne SM, Marmor AK, Madden N, Herrick G. Comprehensive Asthma Management for Underserved 

Children: The San Francisco General Hospital Experience. Paediatric and Perinatal Epidemiology.

Volume 21, Supplement 3, 2007 November; 9-34(6).

6. *Thyne SM, *Navqi, M, Choudhry S, Tsai, H, Ziv E, et al. Ethnic specific differences in bronchodilator 

responsivemess and morbidity among African American, Puerto Rican, and Mexican Asthmatics. *These 

authors contributed equally to this manuscript. Journal of Asthma. 2007 October; 44 (8), 639-48. 

7. Naqvi M, Tsai HJ, Choudhry S, Thyne SM, Navarro D, Nazario S, Rodriguez-Santana JR, Casal J, Torres

A, Chapela R, Watson HG, Meade K, Rodriguez-Cintron W, LeNoir M, Avila PC, and Burchard

8. Orrell-Valente JK, Jarlsberg LG, Tait MA, Thyne SM, Tubash T and Cabana, MD. Parents’ specific 

concerns about daily asthma medications for children. Journal of Asthma, 2007, 44:385-390.

EG. 

Association between IgE levels and asthma severity among African American, Mexican and Puerto Rican 

asthmatics. Journal of Allergy, Asthma and Clinical Immunology. 2007, 121 (1): 137-143.

9. Cabana MD, Abu-Isa H, Thyne SM, Yawn B. Specialty differences in prescribing inhaled corticosteroids for 

children. Clinical Pediatrics, 2007, 46 (8): 698-705.

10. Cabana MD, Chaffin DC, Jarlsberg LG, Thyne SM, and Clark NM. Selective Provision of Asthma SelfManagement Tools to Families. Pediatrics. 2008 April; 121(4):e900-925. 

11. Bergman DA, Sharek PJ, Ekegren K, Thyne SM, Mayer M, Saunders S. The use of telemedicine access to 

schools to facilitate expert assessment of children with asthma. International Journal of Telemedicine and 

Application. 2008 January; 1: 1-7. PMCID: PMC2271044

12. 

. 

Seibold MA, Wang B, Eng C, Kumar G, Beckman KB, Sen S, Choudhry S, Meade K, Lenoir M, Watson 

HG, Thyne S, Williams K, Kumar R, Weiss KB, Grammer LC, Avila PC, Schleimer RP, González Burchard 

E, and Brenner R. An african-specific functional polymorphism in KCNMB1 shows sex-specific association 

with asthma severity. Molecular Genetics, 2008, 17(17):2681-2690. PMCID: PMC2733805

13. 

. 

Naqvi M, Tcheurekdjian H, DeBoard JA, Williams LK, Navarro D, Castro RA, Rodriguez-Santana JR

Chapela R, Watson HG, Meade K, Rodriguez-Cintron W, LeNoir M, Thyne SM, Avila PC, Choudhry S, 

Burchard EG. Inhaled corticosteroids and augmented bronchodilator responsiveness in Latino and African 

American asthmatic patients. Ann Allergy Asthma Immunol. 2008, Jun;100(6):551-7.

14. Tcheurekdijian H, Thyne SM, Williams LK, Via M, Rodriguez-Santana JR, Rodriguez-Cintron W, Avila PC, 

Burchard EG. Augmentation of bronchodilator responsiveness by leukotriene modifiers in Puerto Rican and 

Mexican children. Ann Allergy Asthma Immunol. 2009;102:510-7. 

Biographical Sketches for each listed Senior/Key Person 2 Page 16

Principal Investigator/Program Director (Last, first, middle): McCune, Joseph, M.
15. Seibold MA, Reese TA, Choudhry , Muhammad Salam MT, Beckman K, Eng C, Atakilit A, Meade K, Lenoir

M, H. Watson HG, Thyne S, Kumar R, Weiss KB, Grammer LC, Avila P, Schleimer RP, Fahy JV, RodriguezSantana J, Rodriguez-Cintron W, Boot RG, Sheppard D, Gilliland FD, Locksley RM, and Burchard EG.

Differential Enzymatic Activity of Common Haplotypic Versions of the Human Acidic Mammalian Chitinase 

Protein. J Biol Chem, 2009; 284 (29): 19650-19658. PMCID: PMC2740590 [Available on 2010/7/17].

D. Research Support

Current Research Support

HHSN272200700031C (Chambers, PI) 09/15/07-09/14/12 

NIH/NIAID

Clinical Trial for Community-Acquired Methicillin Resistant Stapylococcus Aureus (CA-MRSA) Infections

The purpose of this contract is to advance the field of clinical management for uncomplicated skin and soft 

tissue infection caused by community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) by 

determining the optimal outpatient treatment strategy within multiethnic, urban underserved pediatric 

communities. 

Role: Co-investigator

R01 HD053408 (Shafer, PI) 12/1/07-11/31/10

NIH/NICHD

Parental Attitudes Toward Confidential STI Services in Teens

The purpose of this grant is to examine parental knowledge and attitudes towards confidential STI health 

services for teens. This research would help investigators to strengthen the role of parents and encourage 

partnerships between health care providers and parents to ultimately improve the health outcomes for teens, 

particularly those from underserved populations. 

Role: Co-investigator

R01 ESO15794 (Gonzalez-Burchard, PI) 07/01/08-06/30/11

NIH/NIEHS

Gene-Environments & Admixture in Latino Americans (GALA 2)

The purpose of this project is to identify genetic, social and environmental risk factors for asthma among 

various Latino subgroups recruited throughout the US. 

Role: Co-investigator

 

 

HL098107 (Boushey, H and Cabana, M) 09/30/09-06/30/15

NIH/NHLBI

Biographical Sketches for each listed Senior/Key Person 2 Page 17

Principal Investigator/Program Director (Last, first, middle): McCune, Joseph, M.
Biographical Sketches for each listed Senior/Key Person 2 Page 18

Principal Investigator/Program Director (Last, first, middle): McCune, Joseph, M.
PHS 398 Cover Page Supplement

1. Project Director / Principal Investigator (PD/PI)

* Title:

* Street1:

* City:

* Country:

Street2:

County/Parish:

* State:

* Zip / Postal Code:

Prefix: * First Name:

Middle Name:

* Last Name:

Suffix:

2. Human Subjects

Clinical Trial?

* Agency-Defined Phase III Clinical Trial?

* First Name:

Middle Name:

* Last Name:

Suffix:

3. Applicant Organization Contact

Person to be contacted on matters involving this application

* Phone Number: Fax Number:

Email:

OMB Number: 0925-0001 

Province:

Prefix:

Joseph

M.

McCune

No Yes

No Yes

Olive

Giovannetti

Research Services Analyst II

3333 California Street

Suite 315

San Francisco

San Francisco

USA: UNITED STATES

CA: California

941186215

	


 		

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Clinical Trial & HESC Page 19

Principal Investigator/Program Director (Last, first, middle): McCune, Joseph, M.
4. Human Embryonic Stem Cells

* Does the proposed project involve human embryonic stem cells?

If the proposed project involves human embryonic stem cells, list below the registration number of the 

specific cell line(s) from the following list: http://stemcells.nih.gov/research/registry/. Or, if a specific 

stem cell line cannot be referenced at this time, please check the box indicating that one from the 

registry will be used: 

Specific stem cell line cannot be referenced at this time. One from the registry will be used.

PHS 398 Cover Page Supplement

No Yes

Cell Line(s):

	


 		

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Clinical Trial & HESC Page 20

Principal Investigator/Program Director (Last, first, middle): McCune, Joseph, M.
PHS 398 Modular Budget, Periods 1 and 2

OMB Number: 0925-0001 

1. 

Start Date: End Date:

Budget Period: 1

* Direct Cost less Consortium F&A

A. Direct Costs

B. Indirect Costs

Consortium F&A

* Total Direct Costs

Indirect Cost Type

Indirect Cost 

Rate (%)

Indirect Cost 

Base ($) * Funds Requested ($)

Cognizant Agency (Agency Name, POC Name and Phone Number)

Indirect Cost Rate Agreement Date Total Indirect Costs

2. 

3. 

4. 

* Funds Requested ($)

04/01/2011 03/31/2012

Sponsored Research _ On Campus 54.5

DHHS, (415) 437-7820

08/28/2009

C. Total Direct and Indirect Costs (A + B) Funds Requested ($)

1. 

Start Date: End Date:

Budget Period: 2

* Direct Cost less Consortium F&A

A. Direct Costs

B. Indirect Costs

Consortium F&A

* Total Direct Costs

Indirect Cost Type

Indirect Cost 

Rate (%)

Indirect Cost 

Base ($) * Funds Requested ($)

Cognizant Agency (Agency Name, POC Name and Phone Number)

Indirect Cost Rate Agreement Date Total Indirect Costs

C. Total Direct and Indirect Costs (A + B) Funds Requested ($)

2. 

3. 

4. 

* Funds Requested ($)

Sponsored Research _ On Campus 54.5

DHHS, (415) 437-7820

08/28/2009

04/01/2012 03/31/2013

	


 		

 !"# 

Modular Budget Page 21

Principal Investigator/Program Director (Last, first, middle): McCune, Joseph, M.
PHS 398 Modular Budget, Periods 3 and 4

1. 

Start Date: End Date:

Budget Period: 3

* Direct Cost less Consortium F&A

A. Direct Costs

B. Indirect Costs

Consortium F&A

* Total Direct Costs

Indirect Cost Type

Indirect Cost 

Rate (%)

Indirect Cost 

Base ($) * Funds Requested ($)

Cognizant Agency (Agency Name, POC Name and Phone Number)

Indirect Cost Rate Agreement Date Total Indirect Costs

C. Total Direct and Indirect Costs (A + B) Funds Requested ($)

2. 

3. 

4. 

1. 

Start Date: End Date:

Budget Period: 4

* Direct Cost less Consortium F&A

A. Direct Costs

B. Indirect Costs

Consortium F&A

* Total Direct Costs

Indirect Cost Type

Indirect Cost 

Rate (%)

Indirect Cost 

Base ($) * Funds Requested ($)

Cognizant Agency (Agency Name, POC Name and Phone Number)

Indirect Cost Rate Agreement Date Total Indirect Costs

C. Total Direct and Indirect Costs (A + B) Funds Requested ($)

2. 

3. 

4. 

* Funds Requested ($)

* Funds Requested ($)

	


 		

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Modular Budget Page 22

Principal Investigator/Program Director (Last, first, middle): McCune, Joseph, M.
PHS 398 Modular Budget, Periods 5 and Cumulative

1. 

Start Date: End Date:

Budget Period: 5

* Direct Cost less Consortium F&A

A. Direct Costs

B. Indirect Costs

Consortium F&A

* Total Direct Costs

Indirect Cost Type

Indirect Cost 

Rate (%)

Indirect Cost 

Base ($) * Funds Requested ($)

Cognizant Agency (Agency Name, POC Name and Phone Number)

Indirect Cost Rate Agreement Date Total Indirect Costs

C. Total Direct and Indirect Costs (A + B) Funds Requested ($)

2. 

3. 

4. 

Cumulative Budget Information

* Funds Requested ($)

*Section A, Total Direct Cost less Consortium F&A for Entire Project Period

1. Total Costs, Entire Project Period

Section A, Total Consortium F&A for Entire Project Period

*Section A, Total Direct Costs for Entire Project Period

*Section B, Total Indirect Costs for Entire Project Period

*Section C, Total Direct and Indirect Costs (A+B) for Entire Project Period

2. Budget Justifications

 $

 $

 $

 $

 $

Personnel Justification

Consortium Justification

Additional Narrative Justification

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Modular Budget Page 23

Principal Investigator/Program Director (Last, first, middle): McCune, Joseph, M.
PERSONNEL JUSTIFICATION

Joseph M. McCune, MD, PhD, Principal Investigator, will devote 0.60 calendar months of his effort to this 

project during Years 01 and 02. Dr. McCune is a Professor of Medicine and the Chief of the Division of 

Experimental Medicine at UCSF. He has had over 28 years of experience in taking care of patients with HIV 

disease, 26 years of which have been devoted to research directly related to understanding the mechanisms of 

pathogenesis of disease. He will be involved in the design, implementation, and interpretation of all aspects of 

this study. 

Shannon Thyne, MD, Co-Investigator, will devote 0.60 calendar months of effort to this project in Years 01 

and 02. Dr. Thyne is an Associate Professor of Clinical Pediatrics and the Medical Director of the Children’s 

Health Center at San Francisco General Hospital. She will assist with the collection of samples from patients 

who have been previously recruited by Dr. Burt. 

Trevor Burt, MD, Assistant Adjunct Professor, will devote 2.40 calendar months of his effort to this project 

during Years 01 and 02. Dr. Burt has worked in Dr. McCune’s lab on and off since 2000, when he first came as 

a Sarnoff Research Fellow from Harvard Medical School (2000-2001). After finishing medical school and 

completing an internship and residency at Children’s Hospital/Harvard, he started to work again in Dr. 

McCune’s lab as a postdoctoral fellow (in 2005). He is now a board-certified pediatrician with sub-specialty 

training in neonatal/perinatal medicine, has extensive clinical expertise in the care of newborns and infants, 

has been involved in recruiting subjects into clinical studies in the past, and is fully familiar with all of the 

techniques to be used in this project.

TBH, Staff Research Associate II, will devote 4.80 calendar months of effort to this project in Years 01 and 

02. This individual will assist Dr. Burt with most of the data collection for this project, including work in Specific 

Aims 1 and 2. Tasks will include sample acquisition, logging, and preparation; analyses conducted by flow 

cytometry, ELISA, and PCR; data collection, analysis, and storage; and quality control measurements.

Personnel Justification Page 24

Principal Investigator/Program Director (Last, first, middle): McCune, Joseph, M.
ADDITIONAL NARRATIVE JUSTIFICATION

So that we can finish this project in two years, it is necessary to budget more resources in FY01 for the work in 

Aims 1 and 2. Thus, more cord blood specimens will be analyzed in Year 01 than in Year 02, and all 

specimens will have been obtained halfway through Year 02.The remaining time period of the grant will be 

used for data analysis. These additional resources in FY01 will cost $25,000 or one additional module.

Additional Justification Page 25

Principal Investigator/Program Director (Last, first, middle): McCune, Joseph, M.
PHS 398 Research Plan

1. Application Type:

From SF 424 (R&R) Cover Page. The response provided on that page, regarding the type of application being submitted, is repeated for your 

reference, as you attach the appropriate sections of the Research Plan.

*Type of Application:

OMB Number: 0925-0001 

2. Specific Aims

3. *Research Strategy

6. Protection of Human Subjects

7. Inclusion of Women and Minorities

8. Targeted/Planned Enrollment Table

9. Inclusion of Children

10. Vertebrate Animals

13. Consortium/Contractual Arrangements

14. Letters of Support

15. Resource Sharing Plan(s)

16. Appendix

1. Introduction to Application 

(for RESUBMISSION or REVISION only)

Human Subjects Sections

2. Research Plan Attachments: 

 Please attach applicable sections of the research plan, below.

Other Research Plan Sections

5. Progress Report Publication List

11. Select Agent Research

12. Multiple PD/PI Leadership Plan

4. Inclusion Enrollment Report

New Resubmission Renewal Continuation Revision

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List of Research Plan Attachments Page 26

Principal Investigator/Program Director (Last, first, middle): McCune, Joseph, M.
SPECIFIC AIMS

The development of the mammalian immune system is typically thought to occur in a linear fashion, from 

immaturity to maturity as a function of antigen exposure. Previous findings in birds and in mice, however, 

indicate that this view is oversimplified. Thus, in these species, the developing immune system appears to be

“layered”1 in a manner that is independent of antigen exposure, beginning as a multilineage fetal system that 

is replaced by an anatomically and biologically distinct multilineage system after birth.2,3 If so, then

developmentally ordered and unique hematopoietic stem/progenitor cells (HSPC) could give rise to distinct 

lymphocyte lineages at different stages of development. 

In ongoing experiments, we have found that such immune system “layering” occurs in humans. Our 

preliminary data show that: (1) the human fetus can mount a vigorous immune response to exogenous 

antigens;4 (2) the human fetal immune response to exogenous antigens can be actively suppressed by 

antigen-specific Tregs;4,5 (3) fetal Tregs are derived from a fetal-specific lineage of T cells; (4) fetal and adult 

HSPCs give rise to distinct populations of T cells; and (5) fetal HSPC-derived T cells show an enhanced 

ability to generate Tregs during thymic maturation and upon exposure to foreign antigens in vitro.

6 These data 

suggest that the human immune system is comprised of two distinct waves: one generated from a “fetal” 

HSPC that exists in utero in the fetal liver and bone marrow, and another generated from a superseding 

“adult” HSPC that resides in the bone marrow at later time points. The former gives rise to an immune system 

that is prone to deliver a tolerogenic response to foregin antigens. The latter gives rise to an immune system 

that is more likely to generate an immunoreactive responses (e.g., one including cytotoxic T cells and 

neutralizing antibodies). 

Given these findings, we hypothesize that physiologic layering of immune system ontogeny leads to a normal 

range in the ratio of fetal- to adult-type T cells at birth, with some neonates exhibiting a higher fraction of fetal 

T cells than others; and that those with a high ratio of fetal/adult T cells will generate predominant Th2 

responses to routine childhood immunizations. 

These hypotheses will be addressed in the experiments of the following Specific Aims: 

Specific Aim 1. To determine the normal range of fetal to adult T cells in the umbilical cord blood 

of the full term neonate. 

In these experiments, comprehensive phenotypic, transcriptional, and functional analyses will be carried 

out on umbilical cord blood (UCB) mononuclear cells from a total of 200 normal full-term deliveries, 

obtained over an 18-month time frame from the Human Cord Blood Bank of the UCSF Clinical and 

Translational Sciences Institute, from Dr. Elizabeth Shpall of the University of Texas M.D. Anderson 

Cancer Center, and as part of a prospective study to be carried out with Dr. Shannon Thyne of the Child 

Health Center at San Francisco General Hospital (SFGH). Naïve T cells in these samples will be studied 

to determine the ratio of fetal/adult T cells (TF/TA) and the relationship of this ratio to naïve T cell function.

Specific Aim 2. To determine whether those full-term neonates with a high ratio of fetal/adult T cells

are more likely to generate a Th2-polarized immune response to routine childhood immunizations.

Under the auspices of an existing protocol that has been approved by the UCSF Committee on Human 

Research protocol and in collaboration with Dr. Shannon Thyne, 50 full-term infants will be followed from 

birth through 12 months. Cord blood samples obtained from each of these newborns will be examined for 

the TF/TA ratio and this ratio will be related to the response of the newborn to hepatitis B vaccination. 

We anticipate that this study will reveal normal variation in the ratio of fetal to adult T cells at birth and that 

such variability in this ratio will be directly related to – and possibly causal of – a Th2 skew that results in a 

poor response to childhood vaccines and a heightened predisposition to childhood infections and to atopic 

disorders. If so, then modalities aimed at changing this ratio more towards the adult lineage at birth may 

provide benefit to a substantial number of newborns.

Specific Aims Page 27

Principal Investigator/Program Director (Last, first, middle): McCune, Joseph, M.
RESEARCH STRATEGY

(a) SIGNIFICANCE The developing human immune system faces a balancing act that must be carefully timed. 

On the one hand, it must tolerate the presence of the surrounding mother and her non-inherited maternal 

alloantigens (NIMA) or otherwise risk the potential of engaging a fatal “graft vs. host” disease. On the other 

hand, novel antigens must be recognized as foreign when encountered after birth, triggering a vigorous 

adaptive immune response (e.g., with cytolytic T cells and neutralizing antigens) against them. Otherwise, the 

newborn will be susceptible to diseases caused by multiple infectious agents. 

In ongoing experiments, we have obtained preliminary data (see below) indicating that this switch from a fetaltype to an adult-type immune response is dependent upon the stage-specific appearance of distinct 

multilineage hematopoietic stem/progenitor cells (HSPC).6 

Nonetheless, infection remains a leading cause of death and morbidity in newborns. Not only are neonates 

susceptible to more severe forms of disease caused by human pathogens such as herpes simplex virus 1, 

respiratory syncytial virus, Bordetella pertussis and Staphylococcus aureus), they are also subject to serious 

infection by microbial entities that are commensal flora in adults. For example, even after implementation of 

intensive screening and prevention practices, the estimated rate of Group B Streptococal sepsis in the first 

week of life is 0.34 per 1000 live births, resulting in 60-70 deaths per year.

Thus, in utero, hematopoiesis in the first and second 

trimester is largely sustained by a fetal-type HSPC that gives rise to tolerogenic Tregs; later (and perhaps as 

early as the third trimester), an adult-type HSPC instead gives rise to immunoreactive T cells. The timing of this 

switch coincides with birth and normally allows the newborn to move from a stance of tolerance to one of active 

defence against all foreign antigens. In this manner, the “immune privileged” aspect of mammalian pregnancy 

is preserved while the ability of the newborn to fight infections is also permitted.

7 In addition to the immediate impact 

of neonatal illness and death, the long-term disability resulting from these infections represents a profound 

public health burden.8 Premature infants, in particular, are predisposed to more severe infections from all 

pathogens and can also succumb to fatal infection by microbes that infrequently cause severe disease in 

adults, such as Staphylococcus epidermidis.9 Compared with adults and older children, newborns produce 

less, and generally less effective, antibody in response to most immunizations. They are also less able to 

generate T cells that mediate effective antimicrobial responses.

10-15 Together, these deficiencies render the 

neonate a vulnerable target for a host of invading pathogens. 

If the switch to an “adult-type” immune system is incomplete or overly slow after birth, two other problems may 

also arise. First, the neonate may respond less well to immunizations provided during the first months of life, 

either generating low levels of an effective response or polarized features of a non-effective response.10,11,16-25

Secondly, those neonates that are most likely to develop atopic disorders after birth are also those who are 

most likely to generate suboptimal (and/or strong Th2-type) response to vaccination.21,26 Since fetal Tregs may 

suppress Th1-type (or other) immune responses to vaccines in a manner that is different than adult Tregs, we 

speculate that strong Th2 polarization of childhood responses to vaccines may in part be due to a higher than 

normal proportion of fetal Tregs at birth. 

In the studies of this proposal, we hypothesize that the immune system “layering” that is necessary for effective 

in utero development and postnatal protection of the human fetus occurs at a dissimilar pace in different 

individuals, predisposing some at birth to less effective immune responses to childhood immunizations.

(b) INNOVATION Previous experiments have demonstrated that similar “layering” of the immune system can 

occur in avian and murine models.1-3 In these species, however, the timing and/or anatomic constraints are 

entirely different. In particular, the murine immune system develops at a markedly different pace than does the 

human immune system, e.g., with very few Tregs detectable until three days after birth27 as compared to the 

late 1st trimester in the human. This study is innovative in two respects: this is the first time that human immune 

system layering has been studied in utero and at birth; in addition, we have identified and validated a set of 

genes that are uniquely expressed in fetal or adult T cells, allowing us to quantitatively and qualitatively study 

the kinetics of the two populations as a function of time. The proposed research has the potential to improve 

prevention (through improved vaccine strategies) and treatment of neonatal infection (by providing a better 

understanding of normal human fetal immune development), and should teach us how the developmental state 

of the fetus and newborn affects their ability to respond to pathogens or vaccines. 

Research Strategy Page 28

Principal Investigator/Program Director (Last, first, middle): McCune, Joseph, M.
(c) RESEARCH PLAN We propose to test the hypothesis that the immune system of the human newborn is 

comprised of two distinct hematopoietic lineages, one derived from a multilineage HSPC that resides in the 

fetal liver and bone marrow, and another from an HSPC that begins to function later in pregnancy and that 

supplants the fetal lineage thereafter. The former lineage is endowed with tolerogenic T cells that allow the 

fetus to co-exist with the mother (including her NIMA and other foreign antigens circulating with her); the latter

lineage is instead comprised of T cells that are more likely to develop effector functions against novel antigens. 

The basis for this hypothesis is found by precedent in avian and murine models as well as by way of our 

largely unpublished Preliminary Data: 

The human fetus can mount a vigorous immune response to exogenous antigens. Although the human 

fetal and neonatal adaptive immune systems are often described as “immature” (i.e., dysfunctional or 

ineffective at mounting a response to antigenic challenge), there is 

substantial evidence that immune responses can develop at or before 

birth in species such as sheep and nonhuman primates (but, 

interestingly, not mice) (reviewed in refs.16 and 28). To determine 

whether human fetal T cells are responsive against alloantigens, fetal 

(~20 g.w.) lymphocytes from spleen or lymph node (LN) were labeled 

with carboxyfluorescein succinimidyl ester (CFSE) and co-cultured with 

irradiated antigen-presenting cells (APCs) that had been isolated from the 

peripheral blood of a single healthy adult donor. After five days 

substantive proliferative responses were observed for both CD4+ and 

CD8+ fetal T cells (Fig. 1). This finding raised the question: if fetal T cells 

respond so vigorously to alloantigens in vitro, do they not also respond to 

NIMA expressed by maternal cells that have moved into fetal LNs in 

utero? 

The human fetal immune response to exogenous antigens can be actively suppressed by antigenspecific Tregs. We recently demonstrated that human fetal secondary lymphoid tissues contain significantly 

higher frequencies of CD4+

CD25high Tregs than those of adults.

4 Because 

Tregs have been shown to regulate maternal immunity to fetal 

alloantigens,29 we reasoned that fetal Tregs might also play a role in 

suppressing fetal immune responses against invading maternal cells. To 

test this hypothesis, fetal LN cultures were either depleted or “mockdepleted” of Tregs before stimulation with self (autologous), maternal, or 

unrelated allogeneic APCs. Depletion of Tregs resulted in a highly 

significant increase in proliferation of CD4+ and CD8+ T cells responding to 

autologous or maternal APCs, but only a 

slight (yet statistically significant) 

increase in proliferation of those 

responding to unrelated APCs (Fig. 2). 

These data indicate that fetal T cells are 

not inherently deficient at responding to maternal alloantigens; rather, they 

are actively suppressed by fetal Tregs.

The frequency of Tregs in peripheral lymphoid organs changes markedly 

during the course of gestation, falling from ~15–20% of total CD4+ T cells at 

12–20 g.w. to ~3–7% at birth.

30 To test the hypothesis that such a change in 

frequency reflects a greater propensity of fetal naive CD4+ and CD8+ T cells 

to differentiate into Tregs in response to stimulation, fetal LN cells were 

depleted of CD25high Tregs and stimulated in vitro. After a five-day primary 

mixed lymphocyte reaction (MLR), a significant fraction of fetal, but not adult, 

CD4+ and CD8+ T cells had divided and upregulated FoxP3 expression to 

high levels (Fig. 3A, B).

Research Strategy Page 29

Principal Investigator/Program Director (Last, first, middle): McCune, Joseph, M.
Fetal Tregs are derived from a fetal-specific lineage of 

T cells. The above studies revealed profound differences 

in function between fetal and adult T cells that had 

otherwise indistinguishable phenotypes. To determine 

whether such differences are intrinsic to the T cell lineages 

found during these stages of ontogeny, CD4+

CD25high

Tregs and CD4+

CD45RA+CCR7+CD27+ naïve T cells from 

fetal and adult samples were sorted with a FACSDiVA (Fig. 

4A). Microarray analysis (Fig. 4B) identified thousands of 

genes whose expression levels in adult and fetal CD4+

naïve T cell populations differed significantly (P<0.05) and 

in a highly consistent manner between donors, including 

NOG, GZMA, and RGS1 were highly expressed (20-55 

fold greater) by fetal cells whereas NAP1L2, NR3C2, and 

SYT4A were highly expressed by adult cells. 

Fetal and adult HSPCs give rise to distinct populations of T cells. In avian and mouse models, there is 

strong evidence that fetal HSPCs give rise to unique subsets of 

lymphocytes that cannot be generated from adult HSPCs and that 

immune system “layering”1 occurs during ontogeny.2,3 To test whether a 

similar situation exists in humans, we performed a series of experiments 

in which fetal HSPCs from fetal liver and BM (18-22 g.w.) and adult BM 

samples were injected directly into the human Thy/Liv organ of the SCIDhu Thy/Liv mouse.31 Following a 7-8 week maturation period, we were 

able to identify mature thymocyte populations derived from each HSPC 

population, based on the expression of a unique HLA type (typically HLAA2 or A3) expressed by the donor (source of HSPCs) but not by the 

recipient thymic implant.31,32 We isolated mature CD3+

CD4+CD8-CD25-

thymocytes from thymic implants injected with fetal liver, fetal BM, or 

adult BM-derived HSPCs by FACS (FACS Aria), and performed 

microarray analysis on each population (Fig. 5A). We found that both 

HSPC populations from fetal liver and BM gave rise to identical 

populations of CD4+ thymocytes on the basis of gene expression, with no 

differentially-expressed genes between them (Fig. 5B). By contrast, adult 

BM-derived HSPCs gave rise to CD4+ thymocytes that showed 

substantial differences in gene expression patterns compared to each population of fetal HSPC-derived 

thymocytes (Fig. 5B: 1243 and 1162 differentially-expressed genes versus fetal liver and fetal BM, 

respectively). These data are consistent with the hypothesis that the developmental stage of HSPCs is in part 

responsible for the differences seen in peripheral T cell compartments in the fetus and adult, and that layering 

occurs during the ontogeny of the human immune system. 

Fetal HSPC-derived T cells show an enhanced ability to 

generate Tregs during thymic maturation and upon 

exposure to foreign antigens in vitro. CD4+

CD25+Foxp3+

Tregs can be generated during thymic maturation or following 

activation of peripheral T cells. Some evidence indicates that 

Tregs may arise from a committed progenitor that is distinct from 

conventional T cell precursors.33 We observed that fetal HSPCderived thymocyte populations contained significantly greater 

frequencies of Tregs than those derived from adult HSPCs (Fig. 

6A). In accordance with what we observed in peripheral fetal 

and adult T cell populations, we also noted that fetal HSPCderived thymocytes were highly responsive to stimulation with 

allogeneic APCs and showed a propensity to differentiate into 

FoxP3+ Tregs (Fig. 6B). 

In sum, the above Preliminary Data indicate that the fetal 

Research Strategy Page 30

Principal Investigator/Program Director (Last, first, middle): McCune, Joseph, M.
immune system is derived from a HSPC that gives rise to tolerogenic Tregs while the adult HSPC is more likely 

to give rise to immunoreactive T effector cells. At this point, we have very little information about the relative 

balance of these two compartments at birth. It is also not clear whether and to what extent variations in this 

balance may impact upon the response of the neonatal immune system to novel antigens, including those 

associated with routine vaccines or with environmental allergens. The experiments of this proposal are 

designed to explore these questions.

Specific Aim 1. To determine the normal range of fetal to adult T cells in the umbilical cord blood of 

neonates at birth

Hypothesis. Physiologic layering of the human immune system during ontogeny leads to a normal range in 

the ratio of fetal to adult-type T cells at the time of birth, with some neonates born with a more tolerogenic 

immune system than others.

Rationale. As described in the above Preliminary Data, the human fetal immune system is poised to generate 

a tolerogenic Treg response upon stimulation, an attribute that is conferred by an HSPC that resides within the 

fetal liver and bone marrow. After birth, bone marrow-derived HSPC give rise instead to immunoreactive T 

cells with a reduced propensity to generate Tregs. Teleologically, such “layering” of the immune system would 

appear to be consistent with, and possibly necessary for, maintenance of the semi-allogeneic state of 

pregnancy and, reciprocally, for the generation of an active immune response against foreign (e.g., infectious 

agents) after birth. Similar stage-specific waves of distinct hematopoietic progenitors have also been described 

in avian and murine models.

1-3 A key question that remains unanswered is the following: is there interindividual variation in the rate at which the fetal-type hematopoietic system is replaced by the adult-type

system over time? In this Aim, we propose to determine whether and to what extent such variability may exist 

at the time of birth. Given known transcripts that uniquely identify tolerogenic fetal T cells (TF) and 

immunoreactive adult T cells (TA), the normal range of these two T cell subpopulations in the umbilical cord 

blood will be determined.

Experimental Approach. Comprehensive phenotypic, transcriptional, and functional analyses will be carried 

out on umbilical cord blood (UCB) mononuclear cells from a total of 200 normal full-term deliveries. Over an 

18-month time frame, 75 of these samples will be obtained on a recharge basis from the Human Cord Blood 

Bank of the UCSF Clinical and Translational Sciences Institute (see attached letter from Dr. William Balke), 75 

will be obtained on a collaborative basis from Dr. Elizabeth Shpall of the University of Texas M.D. Anderson 

Cancer Center (see attached letter), and 50 will be obtained as part of a prospective study to be carried out 

with Dr. Shannon Thyne of the Child Health Center at SFGH (see attached letter). Initial studies will focus on 

naive T cells obtained by a combination of ficoll hypaque gradient enrichment and FACS sorting; excess cells 

will be viably cryopreserved in liquid nitrogen for future experiments that may interrogate other subpopulations 

of cells. The following assays will be carried out: 

1. Phenotypic analysis of T cell populations. The frequency of various T cell populations in the cord blood 

will be analyzed using standard markers of naïve CD4+ T cells (CD45RA+

CD27+CCR7+), memory/effector T 

cells (CD4+

CD45RO+CD95+HLA-DR+), and Tregs (CD4+CD25highFoxP3+CD127low).5

2. 

 Absolute numbers will 

be quantified using TRUcount tubes (BD).

Transcriptional analysis of naïve T cell populations. Phenotypically-pure naïve CD4+ T cells will be 

obtained by sort purification on a FACSAria (BD) and subjected to qRT-PCR assay to detect transcripts 

(transcript specific to cell-type Z denoted as tZ) that are unique to fetal naive T cells (e.g., NOG, GZMA, 

and RGS1; tTF) or to adult naïve T cells (e.g., NAP1L2, NR3C2, and SYT4A; tTA) as well as transcripts for 

house-keeping genes that are equivalently expressed in fetal and adult naive T cells (e.g., the β chain of 

the T cell receptor or HPRT; denoted by tTX). Each transcript will be quantitated in replicate and three 

standardized ratios of fetal/adult T cell transcripts (F/A-T) will be calculated based on the formula (tTF/tTX

(

)/

tTA/tTX) = (tTF/tTA

(RGS1/SYT4A), and the mean of these ratios will be used to represent the fetal/adult T cell ratio (T

). The three ratios will be F/A-T1 (NOG/NAP1L2), F/A-T2 (GZMA/NR3C2), and F/A-T3 

F/TA

3. 

).

Functional analysis of T cell populations. To test whether UCB T cells upregulate FoxP3 and adopt a 

Treg phenotype upon activation with alloantigens,5 naïve CD4+ T cells will be isolated by FACS and 

stimulated with allogeneic adult APCs plus or minus concurrent stimulation with cross-linking antibodies 

against CD3 and CD28. This type of stimulation reliably leads to Treg differentiation from naive CD4+

 TF

(see Figure 3, Preliminary Data). Prior to stimulation, the cells will be labeled with CFSE for determination 

of proliferation. After six days of stimulation, the cells will be harvested and each T cell subpopulation will 

Research Strategy Page 31

Principal Investigator/Program Director (Last, first, middle): McCune, Joseph, M.
be measured for proliferation (CFSE dilution) and for Treg differentiation (FoxP3 upregulation) by flow 

cytometry. Standard types of cytokine response (Th1, Th2, Th17, and Th22) will be measured by cytokine 

production after six days of differentiation in vitro by carrying out intracellular cytokine flow cytometry (CFC) 

for the following cytokines: IL-2, INF-γ, TNF-α, IL-4, IL-17, and IL-22. To test whether cord blood Tregs are 

better able to suppress Th1 vs. Th2 responses, CD4+ naïve T cells will be sort-purified from a healthy adult 

(or cord blood) donor and cultured under Th1 or Th2 conditions in the presence of anti-CD3/anti-CD28. Th1 

and Th2 polarization will be measured by cytokine production after six days of differentiation in vitro. These 

cells will subsequently be labeled with CFSE and cultured in the presence of different quantities of sortpurified CD4+

CD25+ Treg from allogeneic adult blood or cord blood in the presence of anti-CD3/CD28. 

Suppression of Th1 or Th2 cells will be measured by inhibition of proliferation (by CFSE dilution) and 

suppression of cytokine secretion (by cytokine flow cytometry) after a six-day culture period.

Interpretation of Results. The experiments described in this Aim extend observations that we have made in 

human fetal and adult samples to a much larger number of samples of human cord blood. We anticipate that, 

at term, there will be a normative range of fetal- and adult-type Tregs and HSPCs in cord blood, representing 

variable kinetics by which layering of immune system ontogeny proceeds in different individuals. The relative 

frequency of fetal versus adult T cell-specific transcripts (tTF/tTA) will be taken as a measurement of the actual 

fetal/adult T cell ratio (TF/TA). Thus, we will make the assumption that tTF/tTA = TF/TA. In those cases in which 

the fetal/adult T cell ratio (TF/TA) is high, it is predictable that cord blood naïve T cells will be more likely to 

upregulate FoxP3 upon stimulation and that a predominant tolerogenic response to antigen will ensue. 

Potential Problems and Alternative Approaches. Given our existing data sets and the techniques that have 

already been established in the lab, the experiments of this Aim should be relatively straightforward. Though it 

is highly unlikely, it is possible that there will be no significant variability in the TF/TA ratio in full-term newborns. 

This would be an interesting finding, suggesting that the fetal-to-adult T cell transition occurs earlier during the 

third trimester of pregnancy and is complete at birth. Should this be the case, we will shift out attention to 

human premature infants and to nonhuman primates, each of which can be studied during the timeframe of the 

third trimester. 

Specific Aim 2. To determine whether those full-term neonates with a high ratio of fetal/adult T cells are 

more likely to generate a Th2-polarized immune response to routine childhood immunizations.

Hypothesis. Infants with a high ratio of fetal/adult T cells will generate predominant Th2 responses to routine 

childhood vaccinations. 

Rationale. In human neonates, T cell responses are often characterized by deficient Th1 responses,16-19 a 

reduced capacity to induce T cell memory,20 a high frequency of IgE and IgG4 production,21 a skewed Th2 

response,22-24 and even the induction of hyporesponsiveness.25 Such responsiveness to routine childhood 

vaccines has been found to vary within populations, possibly as a result of genetic and/or environmental 

factors26,34,35 and is in part magnified by the formulation of vaccines with the Th2-polarizing adjuvant, alum.36 In

addition, those neonates that are most likely to develop atopic disorders after birth are also those who are most 

likely to generate suboptimal (and/or strong Th2-type) response to vaccination.21,26,37 Since fetal Tregs may 

suppress Th1-type (or other) immune responses to vaccines in a manner that is different than adult Tregs, we 

speculate that strong Th2 polarization of childhood responses to vaccines may in part be due to a higher than 

normal proportion of fetal T cells at birth. 

Recombinant Hepatitis B (HepB) vaccine is routinely given at birth in the United States and provides an ideal 

opportunity to investigate an in vivo response to antigenic stimulation in the newborn. The immune response to 

HepB vaccine is well studied and is characterized by a meager (10%) seroconversion rate with the first dose at 

birth.38 Subsequent vaccine doses are then observed to yield a substantial antibody response.39 The primary 

neonatal CD4+ T cell response to HepB vaccine is characterized by both Th1 and Th2 cytokine production; 

interestingly, however, the HepB-specific memory CD4+ recall response consists of robust Th2 cytokine 

production at one year of age.40 

Experimental Approach. The experiments of this Aim will be carried out under the auspices of a study that 

has been approved by the UCSF Committee on Human Research protocol (H6325-26775. The effects of 

regulatory T cells on the development of the pediatric immune system; McCune PI). In a prospective study

design, 50 pregnant women will be enrolled prior to delivery. Exclusion criteria will include previous/current 

Research Strategy Page 32

Principal Investigator/Program Director (Last, first, middle): McCune, Joseph, M.
HepB infection (HepBsAg+

), other immunomodulatory infections detected by prenatal screening (HepC, HIV), 

or plans for the use of cord blood for alternative purposes (e.g., banking). With assistance from collaborators in 

the SFGH Child Health Center (see attached letter of collaboration from Dr. Shannon Thyne), cord blood will 

be obtained from these deliveries and each of the 50 infants will be followed with blood draws at 6 and 12 

months. In all cases, infants will have received routine childhood immunizations, including those against HepB

at birth, 1-2, and 6-12 months of age. UCB and infant peripheral blood will be processed by ficoll hypaque and 

concurrent purification of CD4+ cells by negative selection (RosetteSep, StemCell). These cells will be tested 

for the following parameters over time:

1. The fraction of TF vs. TA in the umbilical cord blood. This will be carried out using the qRT-PCR-based 

approach described in Aim 1 to determine the ratio of fetal/adult T cells (TF/TA

2. 

). 

Analysis of the cellular immune response against HepB vaccine. Established flow cytometric assays will 

be used to assess the fraction of CD4+

CD25highFoxP3+CD127low Tregs among CD3+CD4+ T cells at each 

time point. These fractions will be converted to absolute numbers using TruCount tube (BD) analysis of 

whole blood counts prior to CD4+ cell isolation, as described.41 In addition to quantitative phenotyping, the 

ability of circulating Tregs to suppress the proliferation of HepB-specific responder CD4+

CD25- T cells will 

be assessed using well-established methods.5,41,42 UCB or peripheral mononuclear cells that have been 

depleted of CD25+ cells (or mock depleted) will be stimulated with (1) polyclonal activators (cross-linking 

antibodies against CD3 and CD28), (2) soluble HepB antigen, and (3) peptides corresponding to HepB. 

Cytokine production in CD4+

3. 

cells will be assessed by CFC, as described above in Aim 1, to determine 

whether antigen-specific stimulation yields a response that is predominantly Th1 or Th2 in type. 

Levels of circulating Immunoglobulin (Ig) isotypes generated against HepB vaccine. Established ELISAs 

will be used to quantitate circulating levels of vaccine-induced IgG1, IgG2, IgG3, IgG4, and IgE relative to 

total Ig. 

Interpretation of Results. This study will relate the ratio of fetal to adult naïve T cells (TF/TA) to a number of 

immune parameters associated with routine childhood immunizations. In particular, it will be of interest to know 

whether a high TF/TA ratio is associated with a higher propensity towards a less effective “immature” response 

(e.g., a Th2-predominant cytokine response to vaccine antigens and the predominance of less-mature, 

antigen-specific IgG1, IgG3, and IgE compared to IgG2). Infants will be studied both cross-sectionally as well 

as prospectively, and we anticipate that the TF/TA ratio and measures of immaturity (especially a bias towards 

a Th2 response) will decrease with age in tandem. The ability of Tregs to suppress antigen-specific responses 

against HepB will also be measured. Given the hypothesis that TF in the newborn generate tolerance to 

antigens they encounter by becoming Tregs, more robust HepB-specific Treg suppression would be expected 

in those children with a higher TF/TA ratio.

Statistical Analysis. These data will be analyzed in consultation with biostatisticians in the Biostatistics 

Consultation Service, associated with the UCSF CTSI (see attached letter of support from Dr. Peter Bacchetti). 

The sample size that has been chosen will detect a 35% difference in cytokine production, with a study power 

of 0.8 and significance level of 0.05. The statistical approach will use an ANCOVA-type linear model approach 

for each outcome variable. Some data may be longitudinal as well as cross-sectional and we will include 

random effects to account for within-subject correlation. We will explore approaches that treat age as 

continuous, in particular, the Laird-Ware repeated measures model. If the measures exhibit strong skewness 

or outlying values, we will attempt to transform the outcome (e.g., log-transformation) to mitigate these issues. 

If this is not adequate, we will compare the groups using non-parametric (e.g., rank based) methods. 

Potential Pitfalls and Alternative Approaches. The primary challenge in this study is that of recruiting and 

retaining 50 patients over an 18-month period. Given the experience of our collaborator, Dr. Shannon Thyne, 

we believe that this goal is attainable. The Birth Center at SFGH delivers 1250-1300 infants yearly, all of whom 

are under the care of the Division of Neonatology, in which Dr. Burt (an MD investigator in the PI’s lab who will 

be working on this study) is an attending physician. He will therefore have the ability to recruit patients (under 

our CHR-approved protocol) as they are admitted to the hospital in labor. Furthermore, in previous studies 

carried out through the Birth Center, members of the McCune lab have successfully recruited similar numbers 

of patients in a period of approximately one year. Approximately 80% of infants born at SFGH receive their 

primary care in the Child Health Center at SFGH. If by three months of recruitment, we are not meeting 

expected goals, this study will be extended to the Birth Center at UCSF, which has a similar delivery rate and 

where we also have CHR approval to recruit. 

Research Strategy Page 33

Principal Investigator/Program Director (Last, first, middle): McCune, Joseph, M.
PROTECTION OF HUMAN SUBJECTS

In Aim 1, human cord blood specimen will be obtained under the auspices of CHR/IRB approval from the 

UCSF CTSI Human Cord Blood Bank (n=75), from Dr. Elizabeth Shpall of UT M.D. Anderson Cancer Center 

(n=75), and from the Child Health Center of San Francisco General Hospital (SFGH) (n=50). The latter 

population will be followed in a longitudinal study in Aim 2 but will be recruited antentally because a cord 

blood collection is required. For this reason, pregnant women will be approached for recruitment and 

consenting purposes, and the children will be followed from birth until 12 months of age. 

a. Human subjects involvement and characteristics

For Aim 2, pregnant women will be approached for recruitment, consent, and the collection of a focused 

medical history. At birth, a cord blood sample will be obtained. From six months to 12 months of age, each 

subject will have a focused medical history and peripheral blood collection every six months. All pregnant 

women with a gestational age greater than 20 weeks are eligible. Exclusion criteria include certain infections 

(e.g., Hepatitis B or C, and HIV) that can affect the study results. There are no exclusion criteria based on 

gender or race.

Pregnant women and children will be involved in this study, as described above, because this study is 

evaluating the development of immune function from birth until 12 months of age. Their involvement will be in 

providing medical history, and acting as guardian of the children involved in decision-making, including 

consent. All aspects of this study will be performed at San Francisco General Hospital, which is a University 

of California, San Francisco affiliated hospital.

b. Sources of materials:

Information and materials collected during this study will be used for this study only. Viably frozen umbilical 

cord blood cells will be kept for further study related to this project involving markers relevant to fetal and 

newborn immunity that may be discovered in the future. This point will be made clear during the consent 

process, and included in written consent forms. All subjects will have cord blood collected at birth and/or 

peripheral blood collected at each study visit. Peripheral blood will be collected by standard venipuncture. 

Cord blood will be collected from the placenta after delivery of the subject. The volume of peripheral blood to 

be collected is 2 mL per kilogram. The volume of cord blood to be collected is about 20-40 mL depending on 

the size and vascularity of the placenta.

 

Demographic and medical data will be collected from all subjects and their parents. Demographic data will 

include age, sex, and race which will be used for data analysis. Medical data will include the presence or 

absence of inflammatory conditions (infections, autoimmune disorders, etc.), atopic conditions (asthma, 

allergic rhinoconjunctivitis, etc.), exposures that can modify inflammatory/atopic conditions (breast feeding, 

tobacco smoke, pets, etc.), and immunization history. Information will be stored in an encrypted (meeting 

mandated IT security standards) password-protected database and will contain subject identifiers such as 

name, address, date of birth, and medical record numbers. This information is necessary to maintain contact 

with the subjects during the full year of the study. Only the primary investigator and members of the research 

team that will contact the subjects will have access to information linked to subject identifiers. Cells isolated 

and stored from collected blood samples will not have any subject identifiers and will be linked to subjects 

with a number generated specifically for this study.

1. Venipuncture: There may be discomfort associated with obtaining peripheral blood via venipuncture. 

Venipuncture also carries the risk of developing a hematoma at the site of the puncture and a <1% risk of 

developing an infection at the site of puncture.

c. Potential risks:

2. Confidentiality: Certain aspects of the subjects’ medical history and demographics will be collected for this 

study. Loss of privacy may lead to problems with insurability or social stigmatization. Also, some of the 

Protection of Human Subjects Page 34

Principal Investigator/Program Director (Last, first, middle): McCune, Joseph, M.
laboratory evaluations being performed may potentially indicate an immune dysfunction. Loss of privacy in 

regards to this information may also lead to problems with insurability.

Adequacy of protection against risks

a. Recruitment and informed consent: For the longitudinal study described in Aims 1 and 2, subjects will be 

recruited from the SFGH Child Health Center. Pregnant women, upon admission to the Child Health Center 

(typically, in early stages of labor), will be asked by their obstetrician if they would be willing to speak with a 

member of the research team regarding this study; if they agree, a research assistant will be immediately 

available to discuss the details of the study. If, after discussion with the research assistant, the parents would 

like their child to participate in this study, informed consent will be obtained. Informed consent forms will be 

signed by the parents, who will be supplied with a copy of the form. All information will be explained in a nontechnical fashion, ample time will be available for the subjects’ parents to ask any questions, and the parents 

will also be asked if they have any specific questions or concerns about the study. Parents will be informed of 

the risks associated with venipuncture and loss of confidentiality of collected information. Since there are 

many Spanish-monolingual patients who deliver at the SFGH Child Health Center, research associates will 

be required to be fluent in English and Spanish. If a fluent Spanish-speaking research associate is not 

available, hospital interpreter services will be used to interpret all aspects of the consent. Consent forms 

translated into Spanish (and approved by the UCSF Committee on Human Research) will be used if subjects 

are Spanish-monolingual, or indicate that this is their preference. Hospital interpreter services will be 

engaged to translate for other non-English or non-Spanish speaking patients who indicate interest in 

participating. The most common other language spoken by patients in the SFGH Child Health Center is 

Mandarin Chinese. CHR-approved consent forms translated into Chinese will also be available.

1. Venipuncture: Blood draws will be coordinated, as much as feasible, with blood draws obtained during 

routine medical care to minimize the number of times venipuncture is performed. Venipuncture will be 

performed via standard techniques and skin preparation (with alcohol) to minimize the risks of developing a 

hematoma or infection at the site of puncture. A topical anesthetic will be made available for use prior to 

venipuncture if desired by the subjects’ parents. Venipuncture will be performed by a phlebotomist, physician 

or nurse who is experienced in drawing blood from small children to minimize risk of discomfort and to 

ensure provision of appropriate distraction techniques for developmental stage. 

b. Protection against risks:

2. Confidentiality: Data will be handled in a confidential manner to prevent loss of privacy. Paper files will be 

kept in a locked cabinet in a secure laboratory. Electronic files will be stored in an encrypted and password 

protected database on secure university server. Only the primary investigator and members of the research 

team who contact the subjects will have access to information linked to subject identifiers. Cells isolated and 

stored from collected blood samples will not have any subject identifiers and will be linked to subjects with a 

number generated specifically for this study.

Protection of Human Subjects Page 35

Principal Investigator/Program Director (Last, first, middle): McCune, Joseph, M.
INCLUSION OF WOMEN AND MINORITIES

This study has a prospective component in Aims 1 and 2 that will recruit 50 children antenatally, and monitor 

them from birth until 12 months of age. It is anticipated that the number of female and male infants recruited 

will be approximately the same. There are no inclusion/exclusion criteria based on sex or race. Subject 

recruitment will occur at San Francisco General Hospital, a UCSF-affiliated hospital that has a patient 

population reflective of the diversity of the general population of San Francisco, and is demonstrated in our 

expected enrollment table (please see Target/Planned Enrollment Table for details).

Women &Minorities Page 36

Principal Investigator/Program Director (Last, first, middle): McCune, Joseph, M.
Targeted/Planned Enrollment Table 

Study Title:

Total Planned Enrollment:

TARGETED/PLANNED ENROLLMENT: Number of Subjects 

Sex/Gender 

Ethnic Category Females Males Total 

Hispanic or Latino 

Not Hispanic or Latino 

Ethnic Category: Total of All Subjects * 

Racial Categories 

American Indian/Alaska Native 

Asian 

Native Hawaiian or Other Pacific Islander 

Black or African American 

White 

Racial Categories: Total of All Subjects * 

* The “Ethnic Category: Total of All Subjects” must be equal to the “Racial Categories: Total of All Subjects.” 

16 200 32 Human immune system layering and the neonatal response to vaccines 200 4060 94 100 32 5

32 4716 16 200 1616 10060 10 47 1001640 100 12080 5

Planned Enrollment Table Page 37

Principal Investigator/Program Director (Last, first, middle): McCune, Joseph, M.
INCLUSION OF CHILDREN

This study focuses primarily on better understanding the immune system of the human fetus and newborn. 

Aims 1 and 2 involve collection of umbilical cord blood from the placentas of 200 newborns, of whom 50 will 

be recruited before birth and then monitored from birth to 12 months of age. This age range has been chosen 

because: (a) the purpose of this study is to investigate the effects of the state of the immune system at birth 

on the response to immunization; (b) such responses should be discernible within one year; and (c) the 

during of the granting period is only two years. The research team has significant expertise in working with 

children and in performing pediatric research studies. Dr. Shannon Thyne is a board-certified pediatrician 

who has been the Medical Director of the SFGH Children’s Health Center since 2001 and who has been 

involved in many research studies emanating from this clinic. Dr. Burt, who will be the primary link with Dr. 

McCune’s lab, is a board-certified pediatrician with sub-specialty training in neonatal/perinatal medicine. He 

has extensive clinical expertise in the care of newborns and infants, and has also been involved in recruiting 

subjects into clinical studies in the past. Dr. McCune, the PI, has over 28 years of experience in clinical 

investigation, with a focus throughout this time on issues related to the fetal/neonatal immune system. 

Phlebotomy will be carried out in an environment tailored to, and with methods appropriate for, the child’s 

developmental stage. A topical anesthetic will be made available for use prior to venipuncture if desired by 

the subjects’ parents. Venipuncture will be performed by a phlebotomist, physician, or nurse who is 

experienced in drawing blood from small children to minimize risk of discomfort and to ensure provision of 

appropriate distraction techniques for developmental stage. 

Children Page 38

Principal Investigator/Program Director (Last, first, middle): McCune, Joseph, M.
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switch in thymic lymphocyte maturation potential occurs at the level of hematopoietic stem cells. Cell 62:863-

874.

4. Michaelsson, J., J.E. Mold, J.M. McCune, and D.F. Nixon. 2006. Regulation of T cell responses in the 

developing human fetus. J Immunol 176:5741-5748.

5. Mold, J., J. Michaelsson, T.D. Burt, M.O. Muench, K.P. Beckerman, M. Busch, T.H. Lee, D.F. Nixon, and 

J.M. McCune. 2008. Maternal alloantigens promote the development of tolerogenic fetal regulatory T cells in 

utero Science 322:1562-1565.

6. Mold, J., S. Venkatasubrahmanyam, T. Burt, J. Rivera, S.A. Galkina, K. Weinberg, A.J. Butte, C.S. 

Stoddart, and J.M. McCune. 2010. Identification of a unique T cell lineage derived from fetal hematopoietic 

stem cells. in preparation 

7. Phares, C.R., R. Lynfield, M.M. Farley, J. Mohle-Boetani, L.H. Harrison, S. Petit, A.S. Craig, W. Schaffner, 

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Epidemiology of invasive group B streptococcal disease in the United States, 1999-2005. JAMA 299:2056-

2065.

8. Koenig, J.M., and W.J. Keenan. 2009. Group B streptococcus and early-onset sepsis in the era of maternal 

prophylaxis. Pediatr Clin North Am 56:689-708, Table of Contents.

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intensive care unit. J Paediatr Child Health 31:387-391.

10. Siegrist, C.A. 2000. Vaccination in the neonatal period and early infancy. Int Rev Immunol 19:195-219.

11. Siegrist, C.A. 2001. Neonatal and early life vaccinology. Vaccine 19:3331-3346.

12. Garcia, A.M., S.A. Fadel, S. Cao, and M. Sarzotti. 2000. T cell immunity in neonates. Immunol Res 22:177-

190.

13. Adkins, B. 2000. Development of neonatal Th1/Th2 function. Int Rev Immunol 19:157-171.

14. Adkins, B. 2005. Neonatal T cell function. J Pediatr Gastroenterol Nutr 40 Suppl 1:S5-7.

15. Zaghouani, H., C.M. Hoeman, and B. Adkins. 2009. Neonatal immunity: faulty T-helpers and the 

shortcomings of dendritic cells. Trends Immunol 30:585-591.

16. Ridge, J.P., E.J. Fuchs, and P. Matzinger. 1996. Neonatal tolerance revisited: turning on newborn T cells 

with dendritic cells. Science 271:1723-1726.

17. Goriely, S., B. Vincart, P. Stordeur, J. Vekemans, F. Willems, M. Goldman, and D. De Wit. 2001. Deficient 

IL-12(p35) gene expression by dendritic cells derived from neonatal monocytes. J Immunol 166:2141-2146.

18. Goriely, S., C. Van Lint, R. Dadkhah, M. Libin, D. De Wit, D. Demonte, F. Willems, and M. Goldman. 2004. 

A defect in nucleosome remodeling prevents IL-12(p35) gene transcription in neonatal dendritic cells. J Exp 

Med 199:1011-1016.

19. White, G.P., E.M. Hollams, S.T. Yerkovich, A. Bosco, B.J. Holt, M.R. Bassami, M. Kusel, P.D. Sly, and 

P.G. Holt. 2006. CpG methylation patterns in the IFNgamma promoter in naive T cells: variations during Th1 

and Th2 differentiation and between atopics and non-atopics. Pediatr Allergy Immunol 17:557-564.

20. Thornton, C.A., J.W. Upham, M.E. Wikstrom, B.J. Holt, G.P. White, M.J. Sharp, P.D. Sly, and P.G. Holt. 

2004. Functional maturation of CD4+CD25+CTLA4+CD45RA+ T regulatory cells in human neonatal T cell 

responses to environmental antigens/allergens. J Immunol 173:3084-3092.

21. Dannemann, A., R. van Ree, M. Kulig, R.L. Bergmann, P. Bauer, J. Forster, I. Guggenmoos-Holzmann, 

R.C. Aalberse, and U. Wahn. 1996. Specific IgE and IgG4 immune responses to tetanus and diphtheria toxoid 

in atopic and nonatopic children during the first two years of life. Int Arch Allergy Immunol 111:262-267.

22. Rowe, J., C. Macaubas, T.M. Monger, B.J. Holt, J. Harvey, J.T. Poolman, P.D. Sly, and P.G. Holt. 2000. 

Antigen-specific responses to diphtheria-tetanus-acellular pertussis vaccine in human infants are initially Th2 

polarized. Infect Immun 68:3873-3877.

23. Ota, M.O., J. Vekemans, S.E. Schlegel-Haueter, K. Fielding, H. Whittle, P.H. Lambert, K.P. McAdam, C.A. 

Siegrist, and A. Marchant. 2004. Hepatitis B immunisation induces higher antibody and memory Th2 

responses in new-borns than in adults. Vaccine 22:511-519.

24. Vekemans, J., M.O. Ota, E.C. Wang, M. Kidd, L.K. Borysiewicz, H. Whittle, K.P. McAdam, G. Morgan, and 

A. Marchant. 2002. T cell responses to vaccines in infants: defective IFNgamma production after oral polio 

vaccination. Clin Exp Immunol 127:495-498.

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25. Belloni, C., P. Orsolini, M. Martinetti, G. Chirico, R.M. Cerbo, G. Comolli, U. Maccarini, C. Barlassina, C. 

Togni, F. Polatti, and et al. 1993. Control of hepatitis B: evaluation of two different vaccinal schedules in 

newborns from HBsAg negative mothers. New Microbiol 16:237-244.

26. Rowe, J., C. Macaubas, T. Monger, B.J. Holt, J. Harvey, J.T. Poolman, R. Loh, P.D. Sly, and P.G. Holt. 

2001. Heterogeneity in diphtheria-tetanus-acellular pertussis vaccine-specific cellular immunity during infancy: 

relationship to variations in the kinetics of postnatal maturation of systemic th1 function. J Infect Dis 184:80-88.

27. Asano, M., M. Toda, N. Sakaguchi, and S. Sakaguchi. 1996. Autoimmune disease as a consequence of 

developmental abnormality of a T cell subpopulation. J Exp Med 184:387-396.

28. Silverstein, A.M. 1964. Ontogeny of the Immune Response. Science 144:1423-1428.

29. Aluvihare, V.R., M. Kallikourdis, and A.G. Betz. 2004. Regulatory T cells mediate maternal tolerance to the 

fetus. Nat Immunol 5:266-271.

30. Takahata, Y., A. Nomura, H. Takada, S. Ohga, K. Furuno, S. Hikino, H. Nakayama, S. Sakaguchi, and T. 

Hara. 2004. CD25+CD4+ T cells in human cord blood: an immunoregulatory subset with naive phenotype and 

specific expression of forkhead box p3 (Foxp3) gene. Exp Hematol 32:622-629.

31. McCune, J.M., R. Namikawa, H. Kaneshima, L.D. Shultz, M. Lieberman, and I.L. Weissman. 1988. The 

SCID-hu mouse: murine model for the analysis of human hematolymphoid differentiation and function. Science

241:1632-1639.

32. Namikawa, R., K.N. Weilbaecher, H. Kaneshima, E.J. Yee, and J.M. McCune. 1990. Long-term human 

hematopoiesis in the SCID-hu mouse. J Exp Med 172:1055-1063.

33. Pennington, D.J., B. Silva-Santos, T. Silberzahn, M. Escorcio-Correia, M.J. Woodward, S.J. Roberts, A.L. 

Smith, P.J. Dyson, and A.C. Hayday. 2006. Early events in the thymus affect the balance of effector and 

regulatory T cells. Nature 444:1073-1077.

34. Marchant, A., V. Appay, M. Van Der Sande, N. Dulphy, C. Liesnard, M. Kidd, S. Kaye, O. Ojuola, G.M. 

Gillespie, A.L. Vargas Cuero, V. Cerundolo, M. Callan, K.P. McAdam, S.L. Rowland-Jones, C. Donner, A.J. 

McMichael, and H. Whittle. 2003. Mature CD8(+) T lymphocyte response to viral infection during fetal life. J 

Clin Invest 111:1747-1755.

35. Brustoski, K., M. Kramer, U. Moller, P.G. Kremsner, and A.J. Luty. 2005. Neonatal and maternal 

immunological responses to conserved epitopes within the DBL-gamma3 chondroitin sulfate A-binding domain 

of Plasmodium falciparum erythrocyte membrane protein 1. Infect Immun 73:7988-7995.

36. Lindblad, E.B. 2004. Aluminium compounds for use in vaccines. Immunol Cell Biol 82:497-505.

37. Sigurs, N., G. Hattevig, B. Kjellman, N.I. Kjellman, L. Nilsson, and B. Bjorksten. 1994. Appearance of atopic 

disease in relation to serum IgE antibodies in children followed up from birth for 4 to 15 years. J Allergy Clin 

Immunol 94:757-763.

38. Belloni, C., G. Chirico, A. Pistorio, P. Orsolini, C. Tinelli, and G. Rondini. 1998. Immunogenicity of hepatitis 

B vaccine in term and preterm infants. Acta Paediatr 87:336-338.

39. Andre, F.E., and A.J. Zuckerman. 1994. Review: protective efficacy of hepatitis B vaccines in neonates. J 

Med Virol 44:144-151.

40. Ota, M.O., J. Vekemans, S.E. Schlegel-Haueter, K. Fielding, M. Sanneh, M. Kidd, M.J. Newport, P. Aaby, 

H. Whittle, P.H. Lambert, K.P. McAdam, C.A. Siegrist, and A. Marchant. 2002. Influence of Mycobacterium 

bovis bacillus Calmette-Guerin on antibody and cytokine responses to human neonatal vaccination. J Immunol

168:919-925.

41. Hartigan-O'Connor, D.J., C. Poon, E. Sinclair, and J.M. McCune. 2007. Human CD4+ regulatory T cells 

express lower levels of the IL-7 receptor alpha chain (CD127), allowing consistent identification and sorting of 

live cells. J Immunol Methods 319:41-52.

42. Hartigan-O'Connor, D.J., K. Abel, and J.M. McCune. 2007. Suppression of SIV-specific CD4+ T cells by 

infant but not adult macaque regulatory T cells: implications for SIV disease progression. J Exp Med 204:2679-

2692.

References Cited Page 40

Principal Investigator/Program Director (Last, first, middle): McCune, Joseph, M.
 

UNIVERSITY OF CALIFORNIA, SAN FRANCISCO 

BERKELEY y DAVIS y IRVINE y LOS ANGELES y RIVERSIDE y SAN DIEGO y SAN FRANCISCO SANTA BARBARA y SANTA CRUZ 

 Shannon Thyne, MD 

San Francisco General Hospital, Department of Pediatrics 

 1001 Potrero Avenue, Mail Stop 6E 

 SAN FRANCISCO, CALIFORNIA 94110 

May 31, 2010 

Joseph M. McCune, MD, PhD 

Division of Experimental Medicine 

San Francisco General Hospital 

University of California, San Francisco 

1001 Potrero Ave, Building 3, Rm. 601 

San Francisco, CA 94110 

Dear Mike, 

I am writing to confirm that I would be delighted to collaborate with you on the research you are proposing in 

your R21 application, entitled “Human immune system layering and the neonatal response to vaccines.” 

Specifically, as Medical Director of Children’s Health Center at San Francisco General Hospital, I will assist with 

collection of samples from patients who have been previously recruited into your vaccine response study. 

As you know, our clinic follows approximately 80% of the children born at SFGH. Given our successes and 

experience with similar follow-up studies in the past, I am confident that we will be able to work together to 

insure excellent study retention and follow-up rates. I will facilitate the process of identifying and collecting 

samples from these patients when they come for their routine follow up visits. I will also educate clinic staff 

about these protocols and monitor compliance with protocol details. These activities will all be carried out in 

accordance with our CHR-approved protocols. 

I look forward to working with you on this exciting and innovative project. 

Sincerely, 

Shannon Thyne, MD 

Associate Professor of Clinical Pediatrics 

Medical Director, Children’s Health Center 

San Francisco General Hospital 

University of California, San Francisco 

Letters of Support Page 41

Principal Investigator/Program Director (Last, first, middle): McCune, Joseph, M.
University of California 

San Francisco

Epidemiology & Biostatistics 

Division of Biostatistics 

185Berry Street, Lobby 5 

Suite 5700 

San Francisco, CA 94107-1762 

May 28, 2010 

J. M. McCune, MD, PhD 

UCSF Box 1234 

Bldg, 3, Rm. 601 

Division of Experimental Medicine 

San Francisco General Hospital 

1001 Potrero Ave. 

San Francisco, CA 94110 

Dear Mike, 

I am writing to say that my colleagues and I of the Biostatistics Unit of the UCSF 

Clinical and Translational Science Institute would be very happy to provide the 

statistical guidance that you need for your R21 proposal (“Human immune system 

layering and the neonatal response to vaccines”). We can advise you about 

experimental design, proper interpretation of resulting data, and the design of 

further studies, as necessary.

It has been a pleasure to work with you in the past. Given the design of the 

exploratory experiments that you wish to perform as well as the relatively large 

number of samples that you will be examining, I am sure that your studies will 

provide much useful information about the issues you wish to address. 

Please let me know when and how I can help with the proposal or with the design 

or analysis of your studies. 

Best wishes, 

Peter Bacchetti, Ph.D. 

Professor of Biostatistics 

Letters of Support Page 42

Principal Investigator/Program Director (Last, first, middle): McCune, Joseph, M.
June 1, 2010 

Joseph M. McCune, MD, PhD 

Division of Experimental Medicine 

San Francisco General Hospital 

an Francisco 

g 3, Rm. 601 

University of California, S

001 Potrero Ave, Buildin

co, CA 94110 

1

San Francis

Dear Mike: 

As Program Director of the UCSF Clinical and Translational Science Institute‐Clinical Research 

Services (CTSI‐CRS), I am delighted to offer the support of the CTSI‐CRS to you and your 

colleagues to pursue research proposed in your R21 application, “Human immune system 

layering and the neonatal response to vaccines”. 

The primary mission of the CTSI‐CRS is to provide first‐rate infrastructure and services for the 

clinical and translational research programs of UCSF investigators. The CRS targets 

investigators with research funding from peer‐reviewed sources such as the National Institutes 

of Health (NIH) and investigators in the early stages of their career development, and is 

supported in part by an NIH Clinical and Translational Science Award (CTSA) grant 

(UL1RR024131) from the National Center for Research Resources. 

For your study needs, CTSI‐CRS nurses collect cord blood samples for the CTSI‐CRS Human 

Cord Blood Bank that allows UCSF investigators to obtain full‐term, de‐identified human cord 

blood specimens for their research. Under the auspices of a protocol approved by the UCSF 

Committee on Human Research, we will enroll with informed consent, women who are 

undergoing an elective C‐section, and then obtain the clamped cord and placenta so that fresh 

human cord blood (typically 25‐ 80 mls) can be withdrawn. We will provide an expedient 

process by which whole cord blood, anti‐coagulated to your specifications, can be delivered to 

you for further processing.  

Per our approved protocol, there will be no patient identifiers sent with the specimen, although 

we will be able to give you information about the gender and ethnicity of the newborn. All of 

this will take place with a fee‐for‐service recharge mechanism, which we anticipate to be 

pproximately $55 per specimen. We anticipate that at least one cord blood sample per week, 

nd potentially more, will be available for your project. 

a

a

Letters of Support Page 43

Principal Investigator/Program Director (Last, first, middle): McCune, Joseph, M.
I am pleased to be able to offer this service to you – and hope that it will enable you to answer 

the important questions that you are addressing. 

Yours sincerely, 

C. William Balke, MD 

rogram Director 

TSI‐Clinical Research Services 

P

C

Letters of Support Page 44

Principal Investigator/Program Director (Last, first, middle): McCune, Joseph, M.
Elizabeth J. Shpall, M.D.

Ashbel Smith Professor of Medicine

Medical Director, Cell Therapy Laboratory

Director, Cord Blood Therapy

Department of Stem Cell Transplantation and Cellular Therapy

Unit 423

1515 Holcombe Boulevard

Houston, Texas 77030

June 2, 2010

Joseph M. McCune, M.D., Ph.D.

Division of Experimental Medicine

San Francisco General Hospital

University of California, San Francisco

1001 Potrero Ave, Building 3, Rm. 601

San Francisco, CA 94110

Dear Mike,

I am writing to say that my colleagues and I would be happy to collaborate with you on your 

analysis of “layering” during the development of the human immune system. For the purposes of 

the work in Aim 1 on your R21 application (“Human immune system layering and the neonatal 

response to vaccines”), we should be able to send you up to more than 100 cryopreserved cord 

blood specimens during the course of the first 18 months of this project. These would be transferred 

to you under the auspices of our existing MTA (MT2009-5827) and corresponding laboratory 

research protocols. Each is likely to have a volume of 25 mls and we routinely find that the viability 

of the cells upon thawing is > 85%.

Given our interests in the use of such cord blood for the purposes of transplantation, the information 

that you gather about the normative ranges of layering at birth will be quite helpful.

I wish you the best of luck with your grant submission. Please let me know when you are ready to 

begin these experiments.

Sincerely,

Elizabeth Shpall, M.D. 

Letters of Support Page 45

Principal Investigator/Program Director (Last, first, middle): McCune, Joseph, M.
RESOURCE SHARING PLAN(S)

The University of California, San Francisco, is committed to the open and timely dissemination of research 

outcomes. Investigators in the proposed activity recognize that promising new methods, technologies, and 

immunological insights may arise during the course of the research. The Investigators are aware of and 

agreed to abide by the principles for sharing research resources as described by NIH in "Principles and 

Guidelines for Recipients of NIH Research Grants and Contracts on Obtaining and Disseminating 

Biomedical Research Programs." The data generated in this grant will be presented at national or 

international conferences and published in a timely fashion. All final peer-reviewed manuscripts that arise 

from this proposal will be submitted to the digital archive PubMed Central.

Resource Sharing Plan Page 46

Principal Investigator/Program Director (Last, first, middle): McCune, Joseph, M.
PHS 398 Checklist

OMB Number: 0925-0001 

1. Application Type:

From SF 424 (R&R) Cover Page. The responses provided on the R&R cover page are repeated here for your reference, as you answer 

the questions that are specific to the PHS398.

* Type of Application:

Federal Identifier: 

2. Change of Investigator / Change of Institution Questions

Change of principal investigator / program director

Name of former principal investigator / program director: 

Change of Grantee Institution

* Name of former institution:

3. Inventions and Patents (For renewal applications only)

* Inventions and Patents:

If the answer is "Yes" then please answer the following:

* Previously Reported: 

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* First Name:

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* Last Name:

Suffix:

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Checklist Page 47

Principal Investigator/Program Director (Last, first, middle): McCune, Joseph, M.
4. * Program Income

If you checked "yes" above (indicating that program income is anticipated), then use the format below to reflect the amount and 

source(s). Otherwise, leave this section blank.

Is program income anticipated during the periods for which the grant support is requested?

*Budget Period *Anticipated Amount ($) *Source(s)

Yes No

5. * Disclosure Permission Statement

If this application does not result in an award, is the Government permitted to disclose the title of your proposed project, and the name, 

address, telephone number and e-mail address of the official signing for the applicant organization, to organizations that may be 

interested in contacting you for further information (e.g., possible collaborations, investment)? 

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Principal Investigator/Program Director (Last, first, middle): McCune, Joseph, M.