Altered Affinity Maturation in Primary Response to (4-hydroxy-3-nitrophenyl) Acetyl (NP) after Autologous Reconstitution of Irradiated C57BL/6 Mice
Developmental Immunology, September
1044-6672
Altered Affinity Maturation in Primary Response to (4-hydroxy-3-nitrophenyl) Acetyl (NP) after Autologous Reconstitution of Irradiated C57BL/6 Mice*
CARL DE TREZ 0
ANNETTE VAN ACKER 0
GEORGETTE VANSANTEN 0
JACQUES URBAIN 0
MARYSE BRAIT 0
0 Universite Libre de Bruxelles, Institut de Biologie et de Me ́decine Mole ́culaires, Laboratory of Animal Physiology , 12 rue des Profs Jeener et Brachet, Gosselies 6041 , Belgium
Immune responses developing in irradiated environment are profoundly altered. The memory antiarsonate response of A/J mice is dominated by a major clonotype encoded by a single gene segment combination called CRIA. In irradiated and autoreconstituted A/J mice, the level of anti-ARS antibodies upon secondary immunization is normal but devoid of CRIA antibodies. The affinity maturation process and the somatic mutation frequency are reduced. Isotype switching and development of germinal centers (GC) are delayed. The primary antibody response of C57BL/6 mice to the hapten (4-hydroxy-3-nitrophenyl) acetyl (NP)-Keyhole Limpet Hemocyanin (KLH) is dominated by antibodies encoded by a family of closely related VH genes associated with the expression of the l1 light chain.We investigated the anti-NP primary response in irradiated and autoreconstituted C57BL/6 mice. We observed some splenic alterations as previously described in the irradiated A/J model. Germinal center reaction is delayed although the extrafollicular foci appearance is unchanged. Irradiated C57BL/6 mice are able to mount a primary anti-NP response dominated by l1 positive antibodies but fail to produce high affinity NP-binding IgGl antibodies. Following a second antigenic challenge, irradiated mice develop enlarged GC and foci. Furthermore, higher affinity NP-binding IgG1 antibodies are detected.
Germinal centers; Affinity maturation; Irradiation; NP system; Lymphopenic environment
INTRODUCTION
The studies of anti-hapten antibody responses show that
humoral immune responses are dynamic processes where
extensive changes in both clonal composition of the
responding B cell population and the structure and
function of the antibodies expressed take place. The
response to the arsonate hapten coupled to Keyhole
Limpet Hemocyanin (KLH) has been well characterized
in A/J mice. Early after a primary immunization with
ARS-KLH, A/J mice produce anti-arsonate antibodies
encoded by several genetic combinations but as the
response proceeds, a major canonical combination,
namely the CRIA idiotype, emerges and dominates the
memory responses
(Manser et al., 1987; Rathbun et al.,
1988)
. This single canonical combination encoded by
VHIDCR11-DFL16.2-JH2/VK10-JK1 is subjected to a
process of hypermutation, allowing the generation and
selection of variants of higher affinity for antigen. The
idiotype CRIA is therefore, a good marker of anti-arsonate
memory response. Immunohistochemical analyses
indicate that splenic ARS þ CRIA þ germinal centers (GC)
and plasma cells are easily detected during the secondary
response in contrast to the primary response (Vora et al.,
1998; 1999; our unpublished observations).
In the course of transfer experiments performed in our
laboratory, we fortuitously discovered that the CRIA
idiotype expression was profoundly altered when the
antiARS response took place in an irradiated environment. For
instance we analyzed irradiated A/J mice (650 rads)
reconstituted with either syngeneic naive spleen cells or
bone marrow cells or irradiated with their hind limbs
partially shielded allowing autoreconstitution. Some
hallmark of memory is retained in these irradiated
recipients: higher concentration of antibodies in the
secondary response. But other hallmarks are lost: the
CRIA idiotype expression and the affinity maturation
(Willems et al., 1990)
. Moreover, the isotype switching
and the development of GC are delayed. Molecular
analysis performed on a set of anti-ARS monoclonal
antibodies established in tertiary response of irradiated
mice shows a reduced frequency of somatic mutation
(Isma¨ıli et al., 1999)
. The primary antibody repertoire
seems frozen even in anamnestic responses.
The primary response to (4-hydroxy-3-nitrophenyl)
acetyl (NP) in C57BL/6 mice has been extensively studied
at both cellular and molecular levels
(Allen et al., 1987;
Lalor et al., 1992; McHeyzer-Williams et al., 1993;
Kelsoe, 1995; Nie et al., 1997)
. At the serum level, NP
binding antibodies are characterized by the expression of
the l1 light chain. High affinity NP binding IgGl are
rapidly detected in the serum (8 days post immunization
with NP-KLH). The heavy chain V regions are encoded by
several germline segments of VH186.2/V3 subfamilly of
the J558 family. A few days after NP-KLH injection,
C57BL/6 mice develop splenic l1þ foci and GC. Around
14 days post-immunization these l1þ histological
structures practically disappear. Among NP reactive
germinal center B cells, the majority expresses BCR
(...truncated)