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Department of Immunology, St. Jude Children’s Research Hospital, Memphis, TN 38105
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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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While B cells effectively recognize the native folded structure of protein Ags, they collaborate with TH cells that recognize composite surfaces of antigenic peptide fragments and MHC class II molecules. Moreover, there is no predictable physical association between B and TH epitopes: B cells specific for influenza hemagglutinin (H)3, for example, can receive help from TH cells reactive with peptides within the hemagglutinin molecule, or from TH cells specific for epitopes within other viral proteins (3). These categorical distinctions between the antigenic structures recognized by B and by TH cells make it conceivable that naive B cells, responding to a novel Ag, might obtain help from TH cells previously primed by encounter with some completely separate antigenic form. That this is more than just a formal possibility is suggested by abundant experimental evidence and supporting theoretical arguments that imply large scale redundancy of recognition among TH cells (4): a given peptide/MHC class II complex can be recognized by a diversity of TCRs, and a given TCR can recognize a diversity of peptide/MHC class II complexes.
If TH cells become irreversibly committed to the secretion of definite patterns of lymphokines, which in turn govern isotype commitment by B cells collaborating with those TH cells, then we may expect the quality of an individual’s Ab response to a newly encountered Ag to depend upon the extent to which naive B cells collaborate with existing memory TH populations, and thus upon the history of that individual’s encounter with immunogens cross-reactive with the new Ag at the TH level. Indeed this is the heart of the "molecular mimicry" theory of immunopathological disease, as it applies to Ab responses (5).
Here we make use of the segmented genetic structure of influenza A viruses to determine the impact upon the quality of a new antiviral Ab response of a memory TH population previously primed by Ag encounter under qualitatively distinct circumstances. Use of the influenza viral strains influenza virus A/HK/X-31 (A/X-31) and influenza virus A/Puerto Rico/8/34 (A/PR8) permits cross-reactivity of TH populations to be separable from B cell specificity: A/X-31 is a reassortant virus, which was constructed in Kilbourne’s laboratory to couple the vigorous growth characteristics of the H1N1 strain A/PR8 with the H3N2 serotype of Hong Kong epidemic influenza viruses (6). Accordingly, A/X-31 inherits six of eight genome segments from A/PR8, and two of eight, those encoding the hemagglutinin and neuraminidase (N) surface glycoproteins that define the serotype, from A/Aichi/2/68 (7). Because MHC class II molecules can be loaded with peptides derived from all viral components, A/X-31 and A/PR8 provoke extensively cross-reactive CD4+ T cell responses (8, 9, 10), and B cells specific for viral glycoproteins can receive help from MHC class II-restricted T cells specific for epitopes within the nucleocapsid core (11). These considerations help explain how, in early studies of heterosubtypic immunity between influenza A viruses, mice that had cleared one viral infection were observed to mount a more rapid and vigorous serum-neutralizing Ab response following challenge with a second serotypically unrelated virus (12). Naive B cells recruited into the primary response to H1 and N1 glycoproteins, following challenge with influenza virus A/PR8 of mice that have recovered from a respiratory infection with A/X-31, will thus receive help from memory TH cells primed in the context of the earlier infection.
We recently demonstrated that the quality of the Ab-forming cell (AFC) response of mice to a respiratory virus can depend critically upon its replicative capacity (13). We now examine the impact of cross-reactive TH populations, primed by pathogenic infection of mice by A/X-31, on the quality of a primary antiglycoprotein AFC response to replicationally inactivated A/PR8.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female C57BL/6J (B6) and B10.BR mice, obtained from The Jackson Laboratory (Bar Harbor, ME) were held under specific pathogen-free conditions until they were used at 10 to 14 wk of age.
Viruses
The origin of influenza viruses A/PR8 and A/X-31 is discussed below. Initial stocks of both strains were obtained from Dr. P. C. Doherty, and viruses were adapted to mice by series of intranasal (i.n.) passages of lung homogenates from infected mice. The A/X-31 stock was passaged five times through mice, and the A/PR8 stock twice, before use. Clarified homogenate stocks, stored at -70°C, had EID50 per milliliter titers as follows (EID50 is the reciprocal of dilution of inoculum required to infected 50% of embryonated chicken eggs): A/PR8, 3.7 x 107; A/X-31, 6.8 x 106. Virus to be used for inactivation or for Ag preparation was grown in the allantoic cavity of embryonated chicken eggs and purified by differential centrifugation and sucrose banding from high titer allantoic fluid stocks. Protein concentrations were determined by the method of Bradford (14). Purified virus in PBS was inactivated by treatment with 0.1% ß-propiolactone (BPL; Sigma, St. Louis, MO), 0.025 M Na2HPO4 at 37°C for 2 h. The mixture was then neutralized with NaHCO3, dialyzed overnight against PBS, and stored at -70°C. Purification of glycoproteins from viral particles was performed as described by Johansson et al. (15).
Immunizations and sampling
Mice were anesthetized with Avertin (2,2,2-tribromoethanol) given i.p. before all immunizations. Infectious and inactivated virus preparations were diluted in PBS, and a 30-µl volume was administered i.n. Mice were euthanized by CO2 inhalation. Superficial cervical and facial lymph nodes (LN) were collected from the cervical region and designated cervical lymph nodes (CLN). The right posterior mediastinal lymph node (MLN) was collected from the posterior thorax. LN nomenclature is based on Tilney (16). LNs were gently disrupted, and single cell suspensions were prepared in IMDM (Whittaker, Walkersville, MD) supplemented with L-glutamine (2 mM), sodium pyruvate (1 mM), penicillin (100 IU/ml), streptomycin (100 µg/ml), gentamicin (10 µg/ml), and 15% FCS (complete medium).
ELISPOT assay
The ELISPOT assay for Ag-specific AFC was done as previously described (17) using nitrocellulose-bottomed 96-well Multiscreen HA filtration plates (Millipore, Bedford, MA) coated with the appropriate viral Ags diluted in PBS, at 1.0 µg/well. After overnight incubation at 4°C, wells were washed and blocked with complete medium. Single cell suspensions were prepared in complete medium, and 100-µl volumes of appropriate dilutions were added to duplicate wells, typically using 1 to 5 x 105 cells/well. Incubation of the plates for 3 to 4 h at 37°C in a humid atmosphere containing 7% CO2 was followed by thorough washing. Alkaline phosphatase-conjugated isotype-specific goat anti-mouse Abs (Southern Biotechnology, Birmingham, AL) diluted 1:500 in PBS plus 5% BSA were added, and the plates were incubated overnight at 4°C. After extensive washing, spots were developed with 1 mg/ml 5-bromo-4-chloro-3-indolyl phosphate (Sigma) in diethanolamine buffer typically for 1–2 h at room temperature, after which the plates were washed and dried. Blue spots reflecting Ab production by individual cells were counted using an Olympus SZH Stereozoom microscope (Olympus Optical, Tokyo, Japan).
It is critical for the correct interpretation of these experiments that
AFC responses against glycoproteins of A/X-31 and A/PR8 be
nonoverlapping, and not include any component directed against the
nucleoplasmid core Ags that are common to both viruses, which could
result from nucleoplasmid contamination of the purified glycoprotein
preparations. Fig. 1
demonstrates that
this was achieved, by comparing the frequency of AFC developed in MLN
of mice infected 7 days previously with A/X-31, scored on filters
coated with glycoproteins prepared from A/X-31 viruses, with
nucleoplasmid cores, or with glycoproteins purified from A/PR8
viruses.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The rationale for these experiments requires the validity of two
premises: 1) that the quality of the AFC response to i.n. infection
with live influenza A virus differs significantly from that elicited by
i.n. immunization with a similar virus preparation rendered inactive in
replication; and 2) that the TH cells initially
recruited into a response to influenza A/X-31 virus can provide
effective help to naive antiglycoprotein-specific B cells activated by
subsequent exposure to influenza A/PR8 virus. The data recovered from
the whole experimental series are comprehensively tabulated, with
statistical quantities, in Table I.
Specific aspects of these data are illustrated and dissected in a more
readily assimilable fashion in the following sections, which will
establish the validity of the premises, and then address the major
issue as to the capacities of memory TH cells.
The statistical data in Table I indicate the considerable
mouse-to-mouse variation encountered in these experiments, as implied
by large, irreducible SDs; it has been necessary to perform these
experiments with fairly large groups of mice to allow confident data
interpretation. Not included in Table I are data from many control
assays that established the high specificity of the AFC reactions: as
demonstrated in Fig. 1
, LN populations from infected mice that
contained large numbers of AFC reactive with glycoproteins isolated
from the infecting virus produced few or no signals when scored on
filters coated with glycoproteins from the heterologous virus, thus
confirming the lack of serological relatedness between A/PR8 and
A/X-31, and the purity of the glycoprotein preparations.
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Fig. 2 compares IgG, IgM, and IgA
antiglycoprotein AFC responses elicited in MLN and in CLN of C57BL/6
mice administered i.n. with influenza viruses A/PR8 or A/X-31 in live
or inactivated forms. (Inactivation was achieved by exposure to the
alkylating agent BPL. In our experience, treatment with BPL, which acts
principally through alkylation of RNA bases (18), results
in complete inhibition of replicative capacity with much less severe
effects on protein structure than do other means of viral inactivation:
complete inactivation can be achieved with little or no impact on H
titer, for example.) As expected from earlier work (13),
responses induced by live viral infection considerably exceed those
induced by inactivated virus (note the difference in histogram scale).
Several other points emerge: 1) infectious and inactivated preparations
elicit antiglycoprotein AFC populations that differ greatly in isotype
distribution. Inactivated A/PR8 and A/X-31 both engender MLN responses
in which switching to IgG is favored over switching to IgA (Fig. 2, A and B), while the reverse is true for CLN
responses, IgA being preferred to IgG (Fig. 2, E and
F). In contrast, administration of either infectious virus
provokes a characteristic early (d7) MLN response featuring
preferential switching to IgA (Fig. 2, C and D),
while IgG is expressed by CLN antiglycoprotein AFC at a frequency
comparable with IgA (Fig. 2, G and H). 2) The CLN
response to A/PR8 infection is almost absent (Fig. 2G); in
fact, the mean values shown in Fig. 2 are due to a small number of mice
that showed a late, breakthrough AFC response in CLN, more typically,
no response was seen. Apart from this difference in the magnitude of
the CLN response to infectious virus, the two viral strains generally
resemble one another in the pattern of the AFC reactions they provoke.
3) The MLN antiglycoprotein AFC response shows a characteristic
evolution during the course of acute infection with either live virus:
the preferential expression of IgA over IgG evident (Fig. 2, C and D) 7 days after inoculation is reversed by
10 days postinoculation (d.p.i.) This evolution in expression of
switched isotypes is not seen in the CLN response to live virus, nor in
antiglycoprotein AFC reactions to inactivated virus at either
site.
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20% of
LD50 dose for C57BL/6 mice) was 200
EID50 of A/X-31, and only 20
EID50 of A/PR8. Clearly, inoculation with such
small numbers of live viral particles must initially result in highly
localized foci of infection, and it is possible that the evident
difference between A/X-31 and A/PR8 in the anatomical distribution of
AFC elicited simply reflects a difference in the distribution of the
sites of initial infection: this is under current investigation.
Fig. 3 shows in greater detail the time
course of the antiglycoprotein AFC response in MLN and CLN to i.n.
infection of C57BL/6 mice with live influenza A/PR8 and A/X-31, with
sampling times 6,7, and 10 d.p.i. and the IgG response specified
by subclass. None of the mice in this experimental group showed any
significant CLN response to infection by A/PR8. The shift in isotype
switch preference between day 7 and day 10 is again evident in MLN AFC
populations. It can be seen that all four IgG subclasses contribute
significantly to the overall IgG response to infection with either of
these influenza viruses, both in MLN and (for A/X-31) in CLN. In this
regard, the C57BL/6 response to infection by these influenza viruses
resembles the response of this IgHb strain to
Sendai virus (SV) infection (13, 19, 20), which was much
less heavily skewed toward IgG2a expression than was seen in the
IgHa mouse strain 129 (19). Other
investigators have pointed out the complexity of the DNA sequence
differences between the IgHa and
IgHb haplotypes, and have suggested that
C
2a genes in the
IgHb haplotype be designated
C2c (21).
Since C2a genes within
all described mouse CH haplotypes function as
true alleles, we have not adopted this suggestion. Here, the roughly
equivalent contribution of all IgG subclasses to the AFC response of
C57BL/6 mice to infection with influenza A/PR8 and A/X-31 viruses
suggests that little information is gained in these experiments by the
dissection of IgG into subclasses. Since pooling IgG subclasses permits
the scoring of significantly more LN cells per assay, it improves the
quality of data recovered when the AFC response is not robust, as when
inactivated virus is the immunogen. Therefore, in all such experiments,
IgG subclasses were not separately enumerated, but were pooled as
overall IgG.
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Given that A/X-31 and A/PR8 influenza viruses elicit extensively
cross-reactive Ag-specific CD4+ T cell
populations, it might be anticipated that the B cell, and AFC, response
to the glycoproteins of A/PR8 of mice that had recovered from infection
with A/X-31 should be enhanced compared with the response of naive
mice. Fig. 4 demonstrates that this is
the case. C57BL/6 mice that had cleared a respiratory infection with
A/X-31 mounted a significantly more robust antiglycoprotein AFC
response to i.n. inactivated A/PR8 than did naive mice. Total AFC
frequencies are severalfold higher 7 d.p.i., both in CLN and in
MLN of A/X-31 immune animals than in naive mice. Moreover, the kinetic
pattern of the response at both sites indicates effective priming by
A/X-31 infection, since the anti-H1/N1 AFC reaction diminished
considerably between 7 d.p.i. and 10 d.p.i. in CLN and MLN of
A/X-31 immune mice while being maintained or increasing at both sites
in naive mice.
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Since the internal, nucleocapsid core structures should be
identical in viruses A/X-31 and A/PR8, mice that have recovered from
infection by A/X-31 are expected to possess primed, memory lymphocytes
specific for nucleocapsid core Ags within both T and B cell subsets.
Such mice should therefore mount a secondary-type AFC reaction against
Ags within the nucleocapsid core when immunized with inactivated A/PR8,
AFC being derived from memory B cell populations. Fig. 5 shows that the anti-nucleocapsid
AFC response to inactivated A/PR8 is very greatly enhanced by earlier
infection with A/X-31, and that it is already dominated by the switched
isotypes IgG and IgA 7 d.p.i. in A/X-31 immune mice.
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The observations reported above constitute the two necessary
premises for determining whether the context of
TH priming must be the major determinant of the
quality of an Ab response. A/X-31 infection primes
TH cells that provide effective help to naive B
cells specific for A/PR8 glycoproteins; and the pattern of isotype
expression among MLN and CLN antiglycoprotein AFC is markedly different
in the response to inactivated A/PR8 particles from that provoked by
infection with live A/PR8. Moreover, the quality of antiglycoprotein
AFC responses provoked by both infectious and inactivated forms of
either virus exhibits a strong dependence on anatomical location that
is particularly marked in the case of inactivated preparations, and is
of an opposite trend for live and inactivated forms. If the
circumstances of the initial recruitment of
TH cells is the primary determinant of the
quality of subsequent immune responses that draw upon those
TH cells, then the preferential expression, in
antiglycoprotein AFC populations provoked in naive mice by i.n.
administration of inactivated A/PR8, of IgG over IgA in MLN, and IgA
over IgG in CLN, should be substantially altered in mice that have
cleared an infection with A/X-31. This experimental plan is shown in
cartoon form in Fig. 6, and relevant data
in Figs. 7 and
8.
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reveal that the pattern of isotype switching
shown by H1/N1-specific B cells in MLN of C57BL/6 mice responding to
inactivated A/PR8 given i.n. is not greatly altered by previous
infection with A/X-31 (compare Fig. 7A with Fig. 2A); isotype switching is considerably skewed toward IgG in
both naive and A/X-31 immune mice, in marked contrast to the initial
IgA skew observed during the response to A/X-31 infection. Switching to
IgA remains relatively much more likely in the less vigorous
antiglycoprotein CLN response to inactivated A/PR8 of A/X-31 immune
mice, as in naive mice, although a higher incidence of AFC-secreting
IgG in A/X-31 immune mice than naive mice may point to a lingering
influence of the earlier infection (compare Fig. 7B with
Fig. 2E). In general, however, the strong regional
discrimination in the quality of the antiglycoprotein AFC response to
inactivated A/PR8 remains unaltered by, and sharply distinguished from,
that of the local AFC reactions characteristically induced by the
earlier A/X-31 infection.
Fig. 8 shows part of the same set of data
in a manner that takes into account the mouse-to-mouse variability
referred to above that is subsumed into the mean values shown in Fig. 7. In Fig. 8, isotype switching tendency for MLN and CLN
antiglycoprotein AFC responses of each individual animal is displayed
as an IgG:IgA ratio, thus independently of magnitude. The same trends
can be discerned: expression of IgA is favored 7 d.p.i. in MLN of
mice infected with A/X-31, while in CLN of the same mice, expression
shows a mild skew toward IgG; trends are reversed in naive mice
administered i.n. with inactivated A/PR8, which show pronounced skews
toward IgG in MLN, IgA in CLN. In this figure, the critical data points
from the "memory" mice, initially infected with live A/X-31 and
subsequently immunized with inactivated A/PR8, can be clearly seen to
fall comfortably into the ranges obtained from naive mice administered
with inactivated A/PR8, and not with infectious A/X-31. Statistical
analysis confirmed the impression of a highly significant difference in
switched isotype expression between antiglycoprotein AFC populations
induced in MLN 7 d.p.i. by inactivated A/PR8 in A/X-31 immune mice
and live A/X-31 in naive mice.
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), contrasting with the secondary-type
response observed in the same mice to Ags within the nucleocapsid core
(Fig. 5). B10.BR mice generally resemble C57BL/6 mice in the influence of heterosubtypic infection on the response to inactivated virus
To ensure that our conclusions were not unduly influenced by some
unsuspected idiosyncrasy in the response of C57BL/6 mice to influenza
A, we performed the same experiments, on a smaller scale, in B10.BR
mice, which are genotypically H2k and thus will
involve a completely separate set of viral epitopes in their
TH response to the virus. The data obtained,
which are listed in Table I but not otherwise graphed, reinforced the
impression derived from the more extensive C57BL/6 experiments. The
response patterns of naive mice to i.n. administration of infectious
A/X-31 and of inactivated A/PR8 recapitulate themes seen in C57BL/6.
There is a strong initial switching preference toward IgA evident in
the day 7 MLN response to infectious A/X-31 that shifts markedly by day
10; and B cells responding to inactivated A/PR8 are much more likely to
switch to IgG in MLN than in CLN. The antiglycoprotein response induced
by inactivated A/PR8 in mice that had recovered from an earlier A/X-31
infection, which was sampled only 7 d.p.i., shows that the prior
infection results in a day 7 MLN and CLN AFC response increased in
magnitude, but unaltered in the pattern of isotype switching, which is
characteristic of that induced in naive mice by inactivated, rather
than live, virus.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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; and TH2 cells, which secrete
preferentially IL-4, IL-5, and IL-10. However, most of the available
information about the biology and phenotypic stability of
TH1 and TH2 cells derives
from studies in tissue culture. This limitation, as well as the
inherent a priori unlikeliness that only two positions should ever be
occupied in the n-dimensional hyperspace theoretically
implied by the capacity of TH cells to express
n variable effector phenotypic quantities, has led some to
doubt whether the TH1/TH2
paradigm allows an accurate description of the physiological behavior
of TH cells in vivo (22, 23). The pattern of isotypes expressed by B cells responding to the initial appearance of Ag is also considered a direct outcome of the circumstances of Ag encounter by naive CD4+ T cells, since isotype commitment by activated B cells is understood to be dictated by the particular mixture of cytokines secreted by the TH cells providing help (1). In mice, production of Ag-specific IgG2a is considered a distinguishing trait of a response dominated by TH1 cells, while IgG1 expression results from TH2 dominance. These arguments are so well established that it has become commonplace to refer to the "TH1" or "TH2" character of an immune response, given only the isotype distribution of the AFC response (2).
Taken together, these two precepts imply that the quality of an Ab response that depends on naive B cells encountering an Ag for the first time must be determined by the conditions under which the TH cells recruited to help that response were initially primed. Unless a stringent correlation is maintained between B and TH recognition of foreign Ags—that is, that TH cross-reactivity is very rare—B cells responding to a newly encountered Ag could be directed into a response entirely mismatched in quality to the the new Ag, by preexisting memory TH cells initially primed under qualitatively distinct circumstances. Mason has recently reviewed the evidence that individual MHC class II-restricted TH cells can react with a diversity of peptide structures (4), pointing out that simple arithmetical considerations of the size of TH populations in vivo and the repertoire of Ag-receptor structures available to them render an absolute specificity of a single TH cell for a single peptide unsustainable; in fact, TH cross-reactivity is common. The experiments described above were designed to discover the effect of TH cross-reactivity on the compartmentalization of antiviral Ab responses.
In an earlier report, we noted that the pattern of isotype expression in the antiviral AFC response elicited in CLN of mice by i.n. deposition of SV was highly dependent on the biological activity of the virus; inactivated SV provoked a response characterized by the almost exclusive expression of IgA, while IgG expression was favored in the more robust response to live, pathogenic SV (13). That such effects can be strictly compartmentalized was evident in that the IgA-dominated anti-SV response provoked in CLN by SV given i.n. was completely unperturbed by the much more vigorous IgG-skewed response at the same site to i.n. infectious influenza virus A/X-31 in mice that had received both viruses simultaneously. Here we have exploited the segmented nature of the influenza virus genome, which permits dissection of B and TH target Ags by the use of reassortant viruses. This feature, coupled with the dependence of the quality of an antiviral AFC response on the biological activity of the virus, has allowed us to investigate the influence of an established memory TH population upon the pattern of a naive B cell response. Will memory TH cells, primed in the exacting context of an immune response to pathogenic respiratory infection with a live virus, imbue naive B cells responding to inactivated virus with the quality of a response to live viral infection; or will the regional character that shapes the B cell response of naive mice to inactivated virus remain the dominant influence?
Data presented above clearly demonstrate the latter result. The antiglycoprotein AFC reaction to i.n. inactivated A/PR8 was considerably larger and faster in mice that had cleared an earlier A/X-31 infection than in naive mice, presumably due to help from cross-reactive TH cells; however, though IgG expression was somewhat more likely in the minor CLN response of A/X-31 immune mice than naive mice, both it and the major MLN response continued to show the marked regional influence characteristic of the reaction in naive mice. AFC reactive with A/PR8 glycoproteins were much more likely to express IgA in CLN than MLN, while MLN AFC were much more likely to express IgG than IgA, trends very distinct from those engendered by live influenza viral infection.
In light of the predominant consensus support for the twin arguments that predict that the circumstances of TH priming should have a profound impact on the quality of subsequent Ab responses—namely, that TH commitment to lymphokine secretion profile is early and stable and that isotype switch choice is directed by TH cytokine concentrations—one must ask why no such impact was evident. One set of answers revolves around the trivial case; namely, that the response to inactivated A/PR8 did not, in fact, include critical TH populations primed by A/X-31 infection. In this regard, it should be noted that primed TH populations can indeed contribute significantly to protective immunity from influenza virus infection by helping Ab responses from naive B cells; cloned TH cells adoptively transferred into histocompatible nude mice enabled recipients to resist lethal viral challenge and to clear the virus (24), an effect not seen after challenge of similarly engrafted SCID recipients, which, lacking B cells, were unable to mount an Ab response. It must be admitted that our experimental design necessarily involves only a partial recruitment of the TH cells primed during the initial infection with live A/X-31; those cells specific for glycoprotein peptides absent from A/PR8 will, of course, not be mobilized by the challenge with the latter viral strain. We doubt that the participation of such clones would result in responses much altered from those reported above for the case of incomplete TH cross reactivity, but direct data is, perforce, lacking, and the individual TH clones specific for determinants within HA, M and NP influenza viral proteins described by Scherle and Gerhard (25), did vary in their ability to support isotype switching.
However, we think rather that the answer to this question lies in the aforementioned promiscuity of TH cells; it seems inescapable that the regulatory mechanisms employed by mammalian immune systems to optimize the matching of Ab class to the type of pathogen encountered, and its route of invasion, evolved in a context of extensive TH cross-reactivity. The powerful nature of the effector clearing mechanisms to which the various Ab classes are differentially linked imposes a stringent need to ensure that pathogen invasion is met by an an Ab response of the appropriate quality. The probability, implied by TH cross-reactivity, that TH cells primed in a response to one type of pathogen would be subsequently recruited into an immune response to a completely different type of pathogen strongly suggests a selection pressure to evolve additional proof-reading measures that can be applied to reinforce an appropriate match of Ab class with pathogen type.
This argument is simply the obverse of that used to support the theory of molecular mimicry as the basis of immunopathological disease; peptide sequences similar to those recognized by T cells activated in response to a diversity of pathogens can readily be found sprinkled throughout the sequences of self, or unrelated foreign, Ags (5, 26). We do not intend to suggest that the T cell response to such peptides never results in immunopathological consequences or qualitatively inappropriate immune responses; only that such undesirable reactions are normally held in check by the extra proof-reading mechanisms here envisaged to have evolved specifically to preclude uncontrolled spillover effects between separate immune responses.
Our earlier observations, particularly of the overriding importance of the site of deposition of inactivated SV for the isotype-switching preference of antiviral B cells in CLN (13), led us to suggest that Ag-transporting dendritic cells (DC) might function to insulate and compartmentalize distinct immune responses within a single node. Since the primary function of DC, by definition, is to activate naive T cells, this compartmental role was most simply envisaged as an interaction, within the draining LN, with Ag-specific CD4+ T cells that recognize viral peptides complexed with MHC class II molecules on the DC surface, and at the same time receive information from the DC about its site of origin and the nature of the Ags carried, which would be used to program the expression of an appropriate pattern of TH cytokines. TH cells thus instructed could then collaborate in a cognate fashion with virus-specific B cells and impose on them a suitable isotype switch preference. The results presented here strongly suggest that the putative instructional role of DC is not encompassed by one quantal event during which TH clonal founders have stamped on them some definite immunoregulatory program that they can execute at another site.
Rather, these new data fit better with the notion that the influence of DC on TH function, while powerful, is limited, certainly in time, and perhaps also in space; TH cells may be imagined as decoding information from DC as to the nature of the Ag they carry and its site of entry, and flexibly "interpreting" this information for Ag-specific B cells in their immediate vicinity. On this theory, alterations in the anatomical distribution of the principal sites of Ag acquisition by DC during the course of a spreading infection might be expected, after some lag, to be reflected in an altered pattern of immune response; we consider it likely that this accounts for the shift in isotype switch preference evident in the MLN antiglycoprotein AFC response between day 7 and day 10 after i.n. infection with live influenza virus. Such a shift would not be expected, and is not seen, during the response to i.n. deposition of inactivated virus, where the much larger initial dose of immunizing Ag would be expected to result in a more anatomically uniform distribution of Ag.
Recent work has convincingly demonstrated significant direct interactions between DC and B cells (27, 28, 29), including the induction by DC, in the absence of T cells, of isotype switching in activated B cells. While T cells, and interaction between TH surface CD40 ligand and B cell surface CD40, are indispensable for effective in vivo class switching by B cells specific for protein Ags, perhaps such direct interactions amplify or modulate cognate TH-B interplay within multicell assemblages during the recruitment phase of the B cell response in LNs, resembling the long-known DC-T-B clusters that can form when these cells are cocultured (30). DC not only very efficiently present TH epitopes complexed with MHC class II molecules, but also have the capacity to retain protein Ags in a native form (27) available to bind specifically to B cell surface Ig. DC appearing in draining LNs may thus be equipped to recruit both naive Ag-specific TH and B cells into such clusters, responding respectively to processed and native forms of the same Ag. In the case of influenza glycoproteins, DC may not require any special means of native Ag retention, since infected DC express large numbers of these proteins, newly synthesized, on their surface, perhaps partially accounting for the high immunogenicity of these viruses, when infectious. Note that DC appear also to be particularly resistant to the cytopathic effects of influenza viruses (31), perhaps lengthening the time for them to initiate immune reactions in draining LN, having been infected at mucosal surfaces. Whatever the nature of the physical association between DC, TH, and B cells, data presented above contradict the idea that the quality of the Ab response to influenza A virus infection of mice is dictated by virus-specific TH clones irrevocably committed only to support responses of a similar quality; the TH clones that help virus-specific B cells make the isotype switches so characteristic of the local response to a live influenza A infection retain the ability to help other B cells make other choices, in response to other DC populations transporting other Ags.
It is certainly not the case that TH cells never
retain any imprint of quality of their priming stimulus. In our own
study of the cytokine-secreting ability of SV-specific
TH cells, we noted that such cells when elicited
by live SV infection secreted much more IFN- upon restimulation in
vitro than similar cells primed by immunization with inactivated SV
(13), and a similar polarization may be reflected in the
increased incidence of IgG among antiglycoprotein AFC induced in CLN by
inactivated A/PR8 in mice that have cleared an A/X-31 infection. Highly
polarized TH cells, irrevocably committed to
TH1 or TH2 secretion
patterns, perhaps by manyfold repeated stimulus (32), may
well underlie immunopathological states. But our observations argue
strongly that such stable commitment is not normally an
immunoregulatory mode of overriding importance, at least in the
response of mice to influenza A infection.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Christopher Coleclough, Department of Immunology, St. Jude Children’s Research Hospital, 332 North Lauderdale Street, Memphis, TN 38105-2794.
3 Abbreviations used in this paper: H, hemagglutinin; AFC, Ab-forming cell; LN, lymph node; CLN, cervical LN; MLN, posterior mediastinal LN; i.n., intranasal(ly); A/PR8, influenza virus A/Puerto Rico/8/34; A/X-31, influenza virus A/HK/X-31; N, neuraminidase; EID50, viral titer as the reciprocal of dilution that will infect 50% of inoculated embryonated chick eggs; BPL, ß-propiolactone; SV, Sendai virus; d.p.i., days postinoculation; DC, dendritic cell; ELISPOT, enzyme-linked immunospot.
Received for publication June 4, 1999. Accepted for publication August 10, 1999.
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antibody to ß2-microglobulin-deficient mice delays influenza virus clearance but does not switch the response to a Th2 phenotype. J. Immunol. 153:1246.[Abstract]
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