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Department of Microbiology and Immunology and the Kimmel Cancer Institute, Thomas Jefferson Medical College, Philadelphia, PA 19107
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recent data have questioned the generality of this model. In an analysis of the primary immune response to vesicular stomatitis virus (VSV), no viral-specific PALS foci were observed, even though virus-specific B cells were routinely found in GC (4). However, the initial stages of this response appear to be T cell independent. Therefore, it remains to be ascertained whether the PALS-associated focus reaction is a necessary step in primary TD B cell proliferation and differentiation, or whether this requirement is limited to immune responses to hapten-protein conjugates.
Another complication in the interpretation of data pertaining to
the relationship between the PALS focus and GC reactions has emerged
from the studies of Klinman’s group (11), who suggested that separate
B cell lineages nucleate early AFC and GC reactions, and give rise to
primary Ab and memory B cell responses, respectively. In this regard,
in the anti-NP response of C57BL/6 mice primary Abs predominantly
bear the 
1 light chain, whereas secondary Abs are predominantly of
the 
isotype (12). In other words, the major clonotypes dominating
the primary anti-NP response do not dominate the anamnestic
responses to this hapten. Not all anti-hapten responses display such a
"repertoire shift." During the primary anti-Ars
(p-azophenylarsonate)-KLH response of A/J mice, a
single anti-Ars clonotype (expressing a single combination of
VH, D, JH, V, and J gene segments, termed
"canonical") becomes predominant, and subsequently continues to
dominate anamnestic responses (13). Since this clonotype fulfills the
criteria of a true "memory clonotype," we investigated the
development of splenic foci and GCs by this and other anti-Ars
clonotypes during the primary anti-Ars-KLH response of A/J
mice.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A/J mice were purchased from The Jackson Laboratories (Bar Harbor, ME). Mice were housed in a specific pathogen-free facility in microisolator cages, and provided autoclaved food and water. Mice (8–10 wk old) were immunized i.p. (100 µg/mouse) with NP13 (Cambridge Research Biochemicals, Cheshire, U.K.)-CGG, Ars8-CGG (Sigma Chemical Co., St. Louis, MO), or Ars-KLH (Calbiochem, La Jolla, CA), all precipitated in alum, for induction of primary responses.
Immunohistochemistry
Spleens were removed at indicated time intervals after immunizations and embedded in Tissue-Tek OCT compound (Fisher Scientific, Bridgewater, NJ) by flash freezing in a 2-methylbutane bath cooled with liquid N2. Frozen spleens were stored at -70°C until sectioned. Six-micron sections were cut on a cryostat microtome, and thaw mounted onto 0.05% polyL-lysine (Sigma Chemical Co.)-coated slides. Sections were air dried, fixed in ice-cold acetone for 10 min, air dried a second time, and stored at -70°C.
Frozen sections were thawed and rehydrated in Tris-buffered saline
(TBS) for 20 min. Endogenous peroxidase activity was blocked by
immersing sections in 0.3% v/v aqueous H2O2
solution. Sections were then blocked with 5% BSA and 0.1% Tween-20 in
TBS and stained with biotinylated mAbs anti-1 Ls136 (10), or
anti-idiotypic Abs E4 or AD8 (14). Ars-BSA-biotin (1.5 µg/ml) was
used for staining Ag-specific cells. NP-CGG-biotin (1 µg/ml) in a
solution containing CGG (10 mg/ml) was used to stain NP-specific cells
in sections derived from mice immunized with NP-CGG. CGG-specific cells
in sections from Ars-CGG-immunized mice were stained with NP-CGG-biotin
(1 µg/ml) only. All slides were incubated with biotinylated Abs or
Ags for 1 h, washed in TBS-BSA, and then incubated with
streptavidin-alkaline phosphatase (Southern Biotechnologies,
Birmingham, AL) for 1 h. PNA (peanut agglutinins) coupled to
horseradish peroxidase (E-Y Laboratories, San Mateo, CA) was used to
identify GCs. Bound alkaline phosphatase and horseradish peroxidase
activities were visualized using Napthol AS-MX/Fast Blue BB and
3-aminoethylcarbazole (Sigma Chemical, St Louis) respectively.
Microdissection of GC, DNA amplification, and sequencing
Idiotope-positive GCs were microdissected from different areas of a stained section obtained from a single spleen using a micromanipulator (Carl Zeiss, Thronwood, NY)-controlled capillary pipette, essentially as described earlier by Jacob and Kelsoe (10). Briefly, scraped tissue containing 50 to 100 cells was placed in tubes containing 15 µl of H2O and 5 µl of PBS. To this mixture, 5 µl of 2 mg/ml proteinase K (Fisher Biotech, Bridgewater, NJ) was added and the mixture was incubated at 37°C overnight. The following day, proteinase K was inactivated by heating at 95°C for 20 min, and then the lysate was subjected to two rounds of PCR amplification. The PCR was carried out using Taq DNA polymerase (Perkin-Elmer Corp., Norwalk, CT) as per the manufacturer’s instructions. Both rounds of PCR contained 40 iterative cycles (95°C for 1 min, 56°C for 30 s, 72°C for 3 min). The primers used for the first round were as follows: 5' VHProm1(5'-GAGCACACTGCTGTCTGACC-3'), which hybridizes to the 5' flanking region of the VH promoter; and 3' JH3–4Int (5'-TCACAAGAGTCCGATAGACC-3'), hybridizing in the intron between JH3 and JH4. Two microliters of the first round products were amplified for 40 additional cycles using the following primer combination: 5' VHProm2EcoRI (5'-GACGAATTCAGTCCTTCCTCTCCAGTT-3'), which is internal to VHProm1; and 3' HindIIIBack (5'-GACTTCAAGCTTCAGTTCTGGC-3'), internal to JH3–4Int. The amplified products were gel purified, digested, and cloned into EcoRI-HindIII-linearized pBluescript vector (Stratagene, La Jolla, CA). The cloned fragments were sequenced using Sequenase (United States Biochemicals, Cleveland, OH) and the internal primers CDR2, hybridizing in the CDR 2 region (5'-GCCCTTGAACTTCTCATTG-3') and JH2–3Int, hybridizing in the intron between JH2 and JH3 (5'CCTAGTCCTTCATGACCTGA-3').
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To investigate the early events associated with the anti-Ars humoral immune response in the spleen, we immunized groups of A/J mice with Ars-KLH in alum, and at different time points processed their spleens for immunohistochemical analysis, as described in Materials and Methods. The majority of clonotypes participating in the primary anti-Ars response express the CRI-A (IdCR) Id. All Abs bearing this IdCR are encoded by a single VH gene segment (VHIdCR) (13). Splenic cryosections were stained for B cells expressing Abs capable of binding Ars-BSA and expressing idiotopes recognized by the anti-IdCR mAbs E4 and AD8. E4 recognizes all somatically mutated and unmutated forms of canonical V regions (14). All Abs encoded by the VHIdCR gene segment in unmutated form are AD8+. A majority of early primary anti-Ars Abs are AD8+ (14).
The earliest Ars-specific B cells observed after immunization were seen
as isolated cells in the red pulp at day 3. Large clusters of
Ars-specific B cells were first detected by day 6 in GCs, and such GCs
continued to be observed through day 14. No PALS-associated foci
specific for Ars or expressing the E4 or the AD8 idiotopes could be
detected at days 3, 6, 9, 12, or 16 of the primary immune response (at
least three mice were examined per time point). In this analysis we
observed more than 60 E4+ and 50 AD8+ GCs,
averaging approximately five independent GCs per spleen from days 6
through 16. A typical staining pattern observed in a splenic
cryosection at day 9 in an Ars-KLH-immunized spleen is shown in Figure 1
. The Ag-specific GC stained with
Ars-BSA (blue) is shown in Figure 1A, and the same GC in
parallel sections stained with anti-idiotypic Abs E4 or AD8 (blue)
is shown in Figure 1, B and C, respectively.
Ars-specific GCs were usually accompanied by scattered Id-positive
cells in adjacent regions of the white and red pulp, some of which were
brightly staining and could be AFCs (Fig. 1, B and
C). The vast majority of the Ars-, E4-, and
AD8-stained cells in the white pulp are confined to the GC,
demonstrating the complete absence of a PALS-associated focus
reaction.
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). Moreover, these heavy chain genes
contained many somatic mutations, consistent with participation of the
resident clonotypes in the B memory cell pathway (data not shown).
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To assess whether the A/J strain had a generic defect in
supporting PALS-associated focus reactions, we immunized groups of mice
with NP-CGG in alum. This particular antigenic preparation had been
shown to support a vigorous splenic focus reaction at the edges of the
PALS in C57BL/6 mice (10). A robust NP-specific splenic focus reaction
was observed in A/J mice during the primary response to NP-CGG, as seen
by staining with NP-CGG-biotin in the presence of excess unlabeled CGG
(Fig. 3A). The
Ag-specific B cells in these foci bore the 1 light chain (Fig. 3B) analogous to what has been reported for the
anti-NP response in C57BL/6 mice. To confirm that these focus
reactions were in the outer regions of the PALS, parallel sections were
stained with an anti-CD4 mAb. As can be seen in Figure 3C, the NP-binding 1+ foci are located at the
edges of the CD4+ T cell-rich PALS region. The intensity of
the staining of the cells in these foci suggests that many were
AFC.
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Earlier work has indicated the requirement of CD4 T cells for the
PALS-associated focus reaction (9). Most studies on the focus reaction
have used the TD carrier protein CGG (9, 10), and it has been proposed
that the molecular form of the Ag can affect the immune response (15).
Therefore, it was of interest to determine whether a different carrier
for the Ars hapten had any effect on the development of the focus
reaction. Since we knew that CGG was compatible with focus formation in
A/J and C57BL/6 mice, we immunized A/J mice with Ars coupled to CGG.
Staining of spleen sections with NP-CGG-biotin revealed the presence of
CGG-specific focus reactions (Fig. 4A) in these mice.
These foci appeared to be composed predominantly of AFC and were
present at the edges of the CD4+ T cell-rich PALS region
(Fig. 4B) as revealed by anti-CD4 staining.
However, E4+ and AD8+ GCs present in the same
histologic section were accompanied by only a few individual
Id+ cells in the adjacent follicles, PALS, and red pulp
(Fig. 4, C and D).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The lack of PALS foci in this response is not due to a generic
deficiency of A/J mice in mounting a focus reaction. A/J mice immunized
with NP-CGG did support a vigorous NP-specific PALS focus reaction,
whose timing of appearance and locale were identical to that observed
in the anti-NP response of C57BL/6 mice (5, 10). Moreover, the
nature of the protein carrier did not influence the focus reaction, as
Ars-specific PALS foci were not observed in A/J mice immunized with
Ars-CGG. However, in these mice, CGG-specific PALS foci could be
readily detected. These CGG-specific PALS foci did not express the 1
light chain, thus ruling out the possibility that focus formation is
restricted to 1-bearing B cell clones (16).
Interestingly, the primary serum anti-Ars Ab response is much
slower to develop than responses to other haptens, such as
phosphorylcholine and NP (17, 18, 19). We found that in A/J mice immunized
with 100 µg of Ars-CGG in alum, no -bearing anti-Ars Abs were
detected in the primary response, and total -bearing anti-Ars Ab
reached a concentration of only 200 to 300 µg/ml by day 13 of this
response. In contrast, A/J mice immunized with 100 µg of NP-CGG in
alum mounted a rapid serum anti-NP response, in which -bearing
Ab reached 600 to 700 µg/ml by day 13. Moreover, -bearing
anti-NP Abs had risen to approximately 5 to 10 times the level of
-bearing anti-NP Abs by this time (data not shown). This high
level of anti-NP serum Ab ( and ) during the early
anti-NP response is correlated with the large PALS-associated AFC
foci present at this time during the response. Conversely, the absence
of an AFC focus reaction during the anti-Ars primary response may
account for the very low serum Ab titers generated during the early
stages of this response. Our results provide a possible explanation for
these observations, in that GC development may be a prerequisite for
AFC formation in the absence of an early PALS focus reaction (20).
Interestingly, anti-Ars AFC cell clusters are observed in the red
pulp during the secondary anti-Ars response (K. Vora and T. Manser,
manuscript in preparation). However, these clusters are far less
compact than the PALS-associated AFC foci observed during the primary
anti-NP response.
A review of previously published data suggests that the absence of focal PALS-associated B cell proliferation and differentiation during the primary immune response may not be unique to the anti-Ars response. Bachmann et al., studying the BALB/c splenic response to VSV, also observed that VSV-specific B cell foci were not induced in the T cell areas but rather in the primary B cell follicles and in the red pulp near the marginal zone (4). Moreover, during the primary immune response of rats to alum precipitated DNP-spider crab hemocyanin, Liu et al. (21) observed only occasional hapten binding B cell blasts in the splenic T cell zone during the first week following immunization, even though DNP-specific B cells did populate GC. These observations are consistent with ours in indicating that initial Ag-driven B cell activation leading to GC formation in normal mice need not take place in the PALS.
Differences in locale of proliferation and differentiation, as well as
migration patterns of B cell clones, could be influenced by many
factors. These include B cell clones arising from distinct lineages
(11), differential dependence on T cell help for priming, site of Ag
localization (3, 4), surface Ig affinity for Ag (22, 23), surface Ig
engagement of self-Ag (24), or prior activation by cross-reactive
foreign Ags (25). Moreover, such factors need not be mutually
exclusive. With regard to these issues, there are certain interesting
similarities and differences between the major B cell clonotypes
responding to NP and Ars in C57BL/6 and A/J strains of mice,
respectively. The "starting affinities" for their cognate epitopes
of the Ag receptors expressed by both these clonotypes
(VH186.2-1 anti-NP and canonical anti-Ars) in
the preimmune repertoire are similar (
Ka of
105 M-1) (26, 27) and hence would not appear
to account for their differential behavior upon Ag stimulation. These
differences in clonotype behavior also could not be easily ascribed to
differences in Ag localization (4), as A/J mice immunized with Ars-CGG
(the anti-NP response was also studied using CGG as carrier) do
form CGG-specific foci while still lacking Ars-specific foci.
Both the predominant anti-NP and anti-Ars clonotypes participate in the primary immune response, but only canonical Ars clones dominate memory responses. This "repertoire shift" in the anti-NP response could be due to the fact that most members of the prevalent primary clonotype terminally differentiate as AFC in PALS-associated foci, and hence cannot dominate memory responses. This repertoire shift is not unique to the NP system. Several laboratories have independently observed that a major clonotype present during the early primary immune response does not dominate memory responses (28, 29, 30, 31, 32). Hence, it is possible that the majority of anti-Ars clonotypes is a member of a memory lineage of B cells, and therefore contributes to serum Ab production only late in primary responses, and during memory humoral responses (11).
Perhaps the most powerful immunoregulation brought to bear on
responding B cells is mediated by CD4 T cells. Therefore, quantitative
and perhaps qualitative differences in the T cell help received by
responding B cell clones might drastically alter their "in situ
behavior." That quantitative differences in T cell help can influence
responding B cell behavior is supported by several previous studies.
During the primary immune responses to the haptens DNP and oxazolone in
carrier-primed animals, Liu et al. (21) observed hapten-specific B cell
foci in the outer layers of the PALS and extending into the red pulp.
These large clusters of B cells were not seen in the primary response
of animals that had not been carrier primed. In contrast, the early
anti-VSV response is T independent, probably explaining the
observation that B cell foci specific for this virus develop in the T
cell-poor red pulp (4). Moreover, Wang et al. observed that during the
immune response to fluorescein-conjugated 
(1—>6) dextran, a
T-independent type-II Ag, large PALS foci did not form, while Ag B
cells could be found in GCs (16).
Even though development of both GC and AFC foci require T cells, the
type and number of T cells required to support these B cell
differentiation pathways are clearly different. Small numbers of
residual and abnormal T cells in nu/nu mice and 
/
T cells in
TCR-/- mice are sufficient to support the GC/memory
pathway in the absence of a detectable AFC response (9, 33). Several
studies on the origin and characteristics of GC CD4 T cells have
indicated that these cells may be functionally distinct from other CD4
T cells (34, 35). Additionally, Kelsoe’s group (36, 37) have shown
that murine splenic GC T cells are immigrants from the PALS, but have
characteristics similar to a novel subset of thymocytes. Our results
suggest that the primary Ars response occurs primarily through the
interaction of Ag B cells with GC T cell help. This clearly questions
the generality of the dogma that all early B cell activation occurs in
a T cell-rich microenvironment (38).
Given all of these considerations, it seems clear that factors both intrinsic and extrinsic to responding B cell clones can influence the microenvironmental locale in which they initially proliferate and differentiate during an immune response. However, the observation in our experiments that similar carrier-specific T cell help to both Ars and NP clones is compatible with both GC (Ars) and AFC/GC (NP) pathways clearly points to intrinsic differences in the nature of the B cell clones that participate in these two responses. One such difference not related to their cognate foreign Ags is the common anti-self (DNA and other Ags) reactivity associated with canonical and many other IdCR anti-Ars clonotypes (39, 40, 41). Such reactivity could result in anti-Ars clones progressing to either a "pre-activated" or "anergized" phenotype after emergence from the immature B cell pool. This prior ligand engagement could alter the subsequent behavior of these clonotypes in TD foreign Ag-driven responses.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Tim Manser, Kimmel Cancer Institute, BLSB 708, 233 S. 10th Street, Philadelphia, PA 19107. E-mail address:
3 Abbreviations used in this paper: GC, germinal center; Ars, p-azophenylarsonate; PALS, periarteriolar lymphoid sheath; AFC, antibody-forming cell; KLH, keyhole limpet hemocyanin; CGG, chicken gamma-globulin; NP, (4-hydroxy-3-nitrophenyl)acetyl; TD, T-dependent; VSV, vesicular stomatitis virus; TBS, Tris-buffered saline; PNA, peanut agglutinin.
Received for publication July 17, 1997. Accepted for publication October 2, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(1—>6) dextran. Proc. Natl. Acad. Sci. USA 91:2502.This article has been cited by other articles:
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V. M. Lentz and T. Manser Cutting Edge: Germinal Centers Can Be Induced in the Absence of T Cells J. Immunol., July 1, 2001; 167(1): 15 - 20. [Abstract] [Full Text] [PDF]
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V. M. Lentz and T. Manser Self-limiting systemic autoimmune disease during reconstitution of T cell-deficient mice with syngeneic T cells: support for a multifaceted role of T cells in the maintenance of peripheral B cell tolerance Int. Immunol., November 1, 2000; 12(11): 1483 - 1497. [Abstract] [Full Text] [PDF]
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A. C. Grammer, R. D. McFarland, J. Heaney, B. F. Darnell, and P. E. Lipsky Expression, Regulation, and Function of B Cell-Expressed CD154 in Germinal Centers J. Immunol., October 15, 1999; 163(8): 4150 - 4159. [Abstract] [Full Text] [PDF]
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K. A. Vora, K. Tumas-Brundage, and T. Manser Contrasting the In Situ Behavior of a Memory B Cell Clone During Primary and Secondary Immune Responses J. Immunol., October 15, 1999; 163(8): 4315 - 4327. [Abstract] [Full Text] [PDF]
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