COMMENTARY

Hemophilia and immunology at the crossroads

P . LOLLARAflac Cancer Center and Blood Disorders Service, Children’s Healthcare of Atlanta, Atlanta, GA and the Department of Pediatrics, Emory

University, Atlanta, GA, USA

To cite this article: Lollar P. Hemophilia and immunology at the crossroads. J Thromb Haemost 2006; 4: 2170–1.

See also Rawle FE, Pratt KP, Labelle A, Weiner HL, Hough C, Lillicrap D. Induction of partial immune tolerance to factor VIII through prior mucosal

exposure to the factor VIII C2 domain. This issue, pp 2172–9.

Her only fault, and that is faults enough, is that she is

intolerable

William Shakespeare, The Taming of the Shrew.

The development of inhibitory antibodies to factor (F) VIII

is the most serious complication in the management of

hemophilia A. Over 30 years ago, Brackmann and co-workers

in Germany, attempting to control the bleeding in a FVIII

inhibitor patient using a prothrombin concentrate and large,

twice-daily i.v. doses of FVIII, unexpectedly observed a 10-fold

decrease in the patient’s inhibitor titer [1]. Experimentation

with this approach led to the development of the Bonn protocol

for immune tolerance induction (ITI) [2]. This and other ITI

regimens have been successful in the permanent eradication of

FVIII inhibitors in most patients in which they have been

attempted [3].

In parallel with the experience of ITI in FVIII inhibitor

patients, the study of tolerance has been a major area of

investigation in immunology. Tolerance, which is defined as the

failure to respond to an antigen, is an active process that

addresses the fundamental problem in vertebrate immunology:

how does the immune system discriminate infectious non-self

from non-infectious self? [4]. From the hematologist’s perspec-

tive, a mechanistic understanding of immune tolerance poten-

tially could replace existing empirical ITI protocols with a

rational, more effective and less expensive therapy. Conversely,

perhaps the immunologist, for whom efforts to treat type 1

diabetes mellitus, multiple sclerosis, rheumatoid arthritis and

other autoimmune diseases by ITI have been largely ineffective

[5], could learn something from the success of ITI in

hemophilia. However, the general study of immune tolerance

and the specific study of ITI in hemophilia have been largely

unconnected. For example, standard textbooks of immunology

do not mention hemophilia when discussing immune tolerance

[6,7].

It is with this background that several groups have began to

study and manipulate the immune response to FVIII, taking

advantage of a murine model of hemophilia A [8] in which

FVIII inhibitors readily develop [9]. Eradication of inhibitors in

hemophilia A mice using parenteral therapy analogous to

human ITI has not been reported. However, in this issue,

Rawle et al. [10] describe an alternative approach of producing

immune tolerance by mucosal, rather than parenteral, delivery

of FVIII. Mucosal tolerance, which initially was described

nearly 100 years ago [11], can be induced by oral or intra-nasal

delivery of antigen. It appears to be a regulatory function of the

immune system that prevents hypersensitivity reactions to food

and microbial antigens by rendering T cells in the periphery

unresponsive to antigen. This could occur through events that

result in functional or actual elimination of the T cell (anergy or

deletion) or in the production of regulatory T cells that produce

tolerance by suppression of naı̈ve T cells [5,12]. In addition to

CD4+CD25+ regulatory T cells, tumor growth factor (TGF)-

b-producing Th3 cells and interleukin (IL)-10-producing Tr1

cells have been implicated in the latter process. Which of these

mechanisms operate is controversial, and may depend on the

antigen and/or the dose of antigen.

Rawle et al. studied the effects of oral and nasal adminis-

tration of purified FVIII C2 domain (FVIII-C2) to hemophilia

Amice prior to a single s.c. injection of FVIII-C2 or full-length

FVIII in incomplete Freund’s adjuvant. Thus, the FVIII

challenge used in this initial proof-of-principle study, differs

from the repeated, i.v. delivery of FVIII that results in clinical

inhibitor formation. The C2 domain was selected because it is

an immunodominant domain in the human FVIII inhibitor

response [13] and because large quantities of recombinant

FVIII-C2 are available for oral administration [14]. FVIII-C2

produced inhibitory antibodies in control mice receiving

mucosal administration of saline, demonstrating that the C2

domain is immunogenic in murine hemophilia A. The average

inhibitor response was reduced approximately 10-fold in mice

receiving either oral or intra-nasal FVIII-C2 prior to challenge

with FVIII-C2. Additionally, mucosal administration of

FVIII-C2 decreased the titer of C2-specific antibodies in mice

challenged with full-length FVIII. However, the FVIII inhib-

Correspondence: Pete Lollar, Room 416D, Emory Children’s Center,

2015 Uppergate Drive, Atlanta, GA 30322, USA.

Tel.: +1 404 727 5569; fax: +1 404 727 4859; e-mail:

jlollar@emory.edu

Journal of Thrombosis and Haemostasis, 4: 2170–2171

� 2006 International Society on Thrombosis and Haemostasis

itor titer was not decreased in this group, indicating that non-

C2 inhibitor epitopes play a major role in the immune response

to FVIII, as is the case in humans. It will be interesting to see if

mucosal administration of full-length FVIII or a mixture of

recombinant FVIII domains in this model can prevent FVIII

inhibitor formation.

Mucosal tolerance induced by FVIII-C2 was associated with

a decrease in the secretion of interferon-c and an increase in the

secretion of IL-10 by FVIII-specific splenocytes from immun-

ized mice. There were no significant differences in IL-2, IL-4,

IL-6 or TGF-b secretion. This result is consistent with the

hypothesis that regulatory T cells express IL-10, which is

involved in the suppression of helper T-cell-dependent humoral

responses. Importantly, adoptive transfer of tolerance to

FVIII-C2 could be obtained in naı̈ve hemophilia A mice using

CD4+ splenocytes frommice treatedwith intranasal FVIII-C2,

further suggesting that regulatory T cells are involved in the

process. However, tolerance was broken by re-challenge with a

single injection of FVIII-C2. This last observation indicates

that considerable work in the hemophilia A mouse model is

required to equal the success currently enjoyed in the clinical

arena. However, further development of the model system

described in the current study and improvements in the

understanding of mechanisms of immune tolerance may

combine to meet this challenge.

Acknowledgements

This work was supported by a grant from the National

Institutes of Health (R01 HL082609, P.L.) and by Hemophilia

of Georgia, Inc.

Disclosure of Conflict of Interests

The author states that he has no conflict of interest.

References

1 Brackmann HH, Oldenburg J, Schwaab R. Immune tolerance for the

treatment of factor VIII inhibitors – twenty years� �Bonn protocol�.VoxSang 1996; 70: 30–5.

2 Brackmann HH, Gormsen J. Massive factor-VIII infusion in haemo-

philiac with factor-VIII inhibitor, high responder. Lancet 1977; ii: 933.

3 DiMichele DM, Kroner BL. The North American Immune Tolerance

Registry: practices, outcomes, outcome predictors. Thromb Haemost

2002; 87: 52–7.

4 Janeway Jr CA. The immune system evolved to discriminate infectious

nonself from noninfectious self. Immunol Today 1992; 13: 11–6.

5 Faria AM, Weiner HL. Oral tolerance. Immunol Rev 2005; 206: 232–

59.

6 Schwartz RH,Mueller DL. Immunological tolerance. In: PaulWE, ed.

Fundamental Immunology. Philadelphia, PA: Lippincott Williams &

Wilkins, 2003: 901–34.

7 Janeway Jr CA, Travers P, Walport M, Shlomchik MJ, eds. Auto-

immunity and Transplantation. Immunobiology. The Immune System in

Health and Disease. New York: Garland Science, 2001.

8 Bi L, Lawler AM, Antonarakis SE, High KA, Gearhart JD, Kazazian

Jr HH. Targeted disruption of the mouse factor VIII gene produces a

model of haemophilia A. Nat Genet 1995; 10: 119–21.

9 Qian J, Borovak M, Bi L, Kazazian Jr HH, Hoyer LW. Inhibitor

antibody development and T cell response to human factor VIII in

murine hemophilia A. Thromb Haemost 1999; 81: 240–4.

10 Rawle FE, Pratt KP, Labelle A, Weiner HL, Hough C, Lillicrap D.

Induction of partial immune tolerance to factor VIII through prior

mucosal exposure to the factor VIII C2 domain. J Thromb Haemost

2006; 4: 2172–9.

11 Wells HG, Osborne TB. The biological reactions of the vegetable

proteins. I: anaphylaxis. J Infect Dis 1911; 8: 66–124.

12 Garside P,Mowat AM, Garside P,Mowat AM. Oral tolerance. Semin

Immunol 2001; 13: 177–85.

13 Prescott R, Nakai H, Saenko EL, Scharrer I, Nilsson IM, Humphries

J, Hurst D, Bray G, Scandella D. The inhibitory antibody response is

more complex in hemophilia A patients than in most nonhemophiliacs

with fVIII autoantibodies. Blood 1997; 89: 3663–71.

14 Pratt KP, Shen BW, Takeshima K, Davie EW, Fujikawa K, Stoddard

BL. Structure of the C2 domain of human factor VIII at 1.5 A reso-

lution. Nature 1999; 402: 439–42.

Hemophilia and immunology at crossroads 2171

� 2006 International Society on Thrombosis and Haemostasis