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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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1:500 for 2 to 3 mo. Thereafter, these
CD4+ T cells tended to distribute more to the spleen than
to the lymph nodes, a pattern that persisted for the life of the
animals. From 3 to 12 mo after infection, virus-specific Thp were
always detectable, although the numbers were diminished relative to
those measured during the acute phase. Thereafter, however, in both
contemporary and cumulative assays, there was a progressive increase in
both the frequency and number of Thp. These increases were especially
apparent for mice more than 2 years of age. This may reflect enrichment
of the CD4+CD44high memory set due to the
gradual diminution of the naive
CD4+CD62LhighCD44low component.
Analysis of DNA staining profiles for the CD4+ T cells
showed high levels of cycling for the acute phase of the response,
whereas the rate of T cell turnover measured for the
CD4+CD44high population by bromodeoxyuridine
incorporation indicated a pattern of stable, continuing proliferation
throughout life. Virus-specific CD4+ T cell memory
resulting from a single exposure to a readily eliminated RNA virus is
thus maintained indefinitely in laboratory mice. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Initial studies of CD4+ T cell memory to Sendai virus used limiting dilution analysis (LDA)3 to determine the frequency of Th cell precursor (Thp) during the first few months after infection (10). Other experiments (11) with the influenza model showed comparable patterns for conventional C57BL/6J (B6) mice and established that CD4+ T cell memory persists for at least 6 mo in congenic, Ig-/- B6.µMT mice (12). Sporadic (or continuing) encounters with Ag-antibody complexes on follicular dendritic cells do not seem to be required for the maintenance of these influenza-specific memory T cells (1). The present kinetic analysis quantitates the acute CD4+ T cell response to Sendai virus and shows that increased numbers of virus-specific Thps are present for the life of a laboratory mouse. The surprising finding is that there is a consistent pattern of enrichment with age.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female B6 mice were purchased from The Jackson Laboratories (Bar Harbor, ME) and, apart from infection with Sendai virus or influenza virus at 8 to 10 wk of age, held under specific pathogen-free conditions (13, 14). The Enders strain of Sendai virus was grown in the allantoic cavity of 10-day-old embryonated hen eggs. Virus stocks were stored at -70°C until use. All mice were anesthetized before intranasal (i.n.) challenge with 30 µl of PBS containing 200 to 500 EID50 of Sendai virus. This range reflects that the mice were primed with different virus stocks over a long interval and makes little difference to the level of Ag challenge, as Sendai virus always replicates to high titers (>107 EID50) in the infected lung (15). All studies of the acute response were done with the 500 EID50 dose. Other 8-wk-old mice were anesthetized and then infected i.n. with 240 hemagglutinating units of the HKx31 influenza A virus (13).
CD4+ T cell enrichment and staining
Single-cell suspensions were made from the cervical or mediastinal lymph nodes (CLN or MLN, respectively) or spleen, the RBC were removed by hypotonic lysis, and the CD4+ T cells were enriched by negative selection or by fluorescence-activated cell sorting (FACS). The CD4+ T cells were negatively selected (11) by exposing spleen or lymph node cells stained with MHC class II (TIB 120) and CD8 (53-6.72) to washed sheep anti-mouse or anti-rat Ig-coated Dynabeads (Dynal, Oslo, Norway) for 40 min at 4°C and then enriched by applying a magnet in accord with the manufacturer’s instructions. Purity (usually 85–95%) was confirmed using a FACScan (Becton Dickinson, Sunnyvale, CA) subsequent to staining with the phycoerythrin-labeled RM-4-5 mAb to CD4. Alternatively, purified CD4+ T cells were sorted to 96 to 99% purity in a FACStarPlus after staining with RM-4-5. In some experiments the cells were additionally stained with biotinylated or FITC-conjugated mAbs to CD44 (IM7) or CD62L (Mel 14), either for phenotyping in two- or three-color mode or for sorting in two-color mode. Biotinylated mAbs were developed with streptavidin red 670 conjugates (Life Technologies, Gaithersburg, MD). All conjugated mAbs were purchased from PharMingen (San Diego, CA) and are referenced in their current catalog.
Thp frequency analysis by IL-2 production
All assays (10, 11) were performed in SMEM (Life
Technologies) supplemented with essential and nonessential MEM amino
acids, sodium pyruvate, L-glutamine, D-glucose,
sodium bicarbonate, 2-ME, gentamicin, penicillin G, streptomycin
sulfate (Sigma, St. Louis, MO), and 10% FCS (Atlanta Biologics,
Atlanta, GA). Enriched or sorted CD4+ T cells were cultured
at a range of densities in round-bottom 96-well plates with 5 x
104 uninfected (NAPC) or virus-infected (VAPC) T
cell-depleted, irradiated (2500 rad) spleen cells in 200 µl of
medium. The T cells were first removed from the APC populations by
treatment with anti-Thy 1.2 mAb (AT83) followed by complement
lysis. The microcultures were incubated for 4 days at 37°C in 10%
CO2, after which 50-µl aliquots of the supernatant were
transferred to new plates. The level of IL-2 activity for each well was
then measured using the CTLL indicator cell line. Values greater than
three times the SD of the mean for medium alone were recorded as
positive. Assay of the medium from responders cultured in the absence
of APCs, or APCs without T cells, gave results similar to the medium
control. Sendai virus-specific Thp frequencies (7, 8) were then
calculated by applying the Poisson formula, in which Fr
= (ur/rl) x
e-u, where Fr is the probability of
obtaining Ag-specific T cells in a microculture when the average Thp
number per well is µ at a given concentration. The fraction of
negatives per total number of wells is given by
F0 = e-u, when
u = 1 and F0 = 0.37.
Theoretically, when the average count for responding T cells per
microculture is 1, 37% of the wells will be scored as negative.
Extrapolation to this point in the LDA gives a number, the reciprocal
of which represents the frequency of Ag-specific cells. The statistical
accuracy of the estimates was determined by 
2 analysis
to give 95% confidence intervals.
IFN-
ELISPOT assay for virus-specific Thp
Multiscreen-HA 96-well filter plates (Millipore, Bedford,
MA) were coated with 4 µg/ml rat anti-mouse IFN- Ab
(PharMingen). Enriched CD4+ T cells obtained from immune
mice at various times after infection were cultured for 72 h with
5 x 105 uninfected or Sendai virus-infected whole
naive spleen cells per well in 200 µl of medium. After culture, the
cells were washed out with PBS containing 0.05% Tween-20, 2
µg/ml biotinylated rat-anti-mouse IFN- Ab (PharMingen) was
added, and the plates were incubated overnight at 4°C. The plates
were again washed with PBS/Tween and then developed with goat
anti-biotin peroxidase (Vector Laboratories, Burlingame, CA) and
3-amino-9-ethyl-carbazole (Sigma) in dimethyl formamide (Fisher,
Norcross, GA). The spots in each well were counted under a microscope,
and the values are expressed as the reciprocal of the frequency of
spot-forming cells relative to the number of CD4+ cells
added to each well at the start of the culture.
Staining for cycling T cells in vitro
After blocking with normal mouse serum at 4°C, single-cell suspensions of lymphocytes were stained with FITC-conjugated anti-CD4 (RM-4-5) or anti-CD8 (53-6.7) and resuspended in PBS containing EDTA (0.087 g/L) and glucose (1.1 g/L). The cells were fixed in ice-cold 95% ethanol and then washed in PBS and stained with the DNA intercalating dye propidium iodide (PI, Sigma) in Nonidet P-40 solution (16). Fluorescence was measured on the FACScan, and the FITC-positive CD4+ or CD8+ T cells were selected and analyzed (16) using CellQuest and ModFit Software (Becton Dickinson).
In vivo cell cycle analysis
Animals were fed water containing 0.8 mg/ml bromodeoxyuridine (BrdUrd, Boehringer Mannheim, Mannheim, Germany) for 5 days (17). The lymphocytes were surface stained (18) for CD4, CD8, CD44, and CD62L by the methods described above. Incorporation of BrdUrd into the DNA of cycling cells was assessed as described by Tough and Sprent (17). Briefly, the cells were fixed and permeabilized with cold 95% ethanol and detergent. The DNA was partially digested with DNase (DN25) and stained with anti-BrdUrd-FITC mAb (Becton Dickinson). Stained cells were analyzed on a FACScan using CellQuest software.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Evidence of a significant Sendai virus-specific Thp response was
apparent in the CLN and MLN as early as day 6 after infection and in
the spleen from day 7 (Table I
). Although
variable, the frequencies of virus-specific CD4+ T cells
reached 1:100 or more in all sites sampled between days 7 and 15 and
were generally >1:400 from day 8 to day 15. Estimating the numbers of
virus-specific CD4+ T cells (Fig. 1A) indicated that (in terms
of total counts) the virus-specific Thp response peaked at about 10
days after infection. Sendai virus cannot be recovered from the lymph
nodes or spleen at any stage and is generally no longer detectable in
the respiratory tract at this time point, although lung titers are
maximal 2 to 3 days earlier (11, 14). Elimination of the virus
between days 8 and 10 is coincident with peak CD8+ CTL
effector function in the lymphocyte population recovered by
bronchoalveolar lavage.
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) in the
CLN, MLN, or spleen between 6 and 9 days after infection (Fig. 2). This
is much more than could be accounted for by LDA (Table I
, Fig. 1A). The discrepancy likely indicates that the LDA is
underestimating the actual magnitude of virus-specific CD4+
T cell response. Nevertheless, the relative kinetics of the CD4 T cell
response are probably not very much different from that shown by the
IL-2 LDA. More accurate measurements will not be possible without
Ag-specific TCR probes, such as MHC tetramers, which are available for
class I- but not class II-restricted T cells (18, 19, 20).
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Long term Thp response profiles were thus analyzed for mice primed
at various times and assayed on the same (Tables
II and III)
or on different (Fig. 1) days. The overall pattern is that the majority
of the virus-specific CD4+ T cells are found in the spleen,
not the lymph nodes, from day 10 to more than 2 years after infection.
This distribution profile is primarily a consequence of the difference
in cell counts between the lymph nodes and spleen (Fig. 1). However, in
a contemporary comparison of mice infected from 2 to 27 mo previously,
the Thp frequencies in the MLN were consistently lower than those in
the spleen (Expt. 2, Table II). The same, general effect was seen for
B6 mice primed up to 6 mo previously with the HKx31 influenza A virus,
although the converse was true for the Ig-/-
B6.µMT mice that have no germinal centers and very small
spleens (11).
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and 4) that contains all evidence of Thp
memory (Expts. 1 and 2, Table IV). This
relative increase in the
CD4+CD62LlowCD44high set (Fig. 3)
is, however, as apparent for the MLN and CLN as for the spleen (Fig. 4). Furthermore, by 12 mo after infection, many of the
CD4+CD44high memory T cells were also
CD62Lhigh (Expt. 3, Table IV). The tendency for
virus-specific memory CD4+ T cells to locate preferentially
to the spleen in the long term is thus not obviously a consequence of
CD62L-mediated recirculation profiles.
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The analysis of Thp frequency profiles showed a tendency for
virus-specific CD4+ T cells to be more prevalent soon after
priming (Table I; Fig. 1A) and in very old mice (Fig. 1B; Expts. 1 and 2, Tables II and III). The former was
expected (1), but the latter was surprising and has not (to our
knowledge) been reported previously. While there was a fair degree of
variability in the frequencies and calculated numbers of virus-specific
CD4+ Thps measured among different individual mice and
different experiments, the trend for the number and frequency to be
lower in middle aged mice and higher in older animals was consistently
observed (Fig. 1B; Expts. 1 and 2, Tables II and III), even
in the experiments on sorted cells (Table IV). This was not unique to
IL-2-producing clones, since estimation of IFN- producing Thp by
ELISPOT assay gave a similar pattern (Table III and 24 . One
possible explanation for this apparent late increase in Thp numbers
could be that CD4+ T cells from older mice may lose
specificity for the priming virus. We found, however, that the capacity
to discriminate between an influenza A virus and a Sendai virus was
retained for mice primed 26 mo previously (Table V).
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All the Sendai virus-specific CD4+ T cells are in the
CD44high set (Expt. 1, Table IV), which becomes the
predominant CD4+ population in older mice (Figs. 3 and 4).
We checked the possibility that CD4+ T cells may tend to
cycle at a higher rate with age, but we found that there was no
difference in BrdUrd incorporation between 2- and 20-month-old mice
(Fig. 5) that were given this thymidine
analogue in their drinking water. The most likely explanation for the
late enrichment of virus-specific CD4+ T cells would seem
to be that these clones are very long lived and become much more
prominent as the magnitude of the naive T cell compartment diminishes
subsequent to thymic involution.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The mechanisms that maintain the size of the Thp pool are not understood (2). The slight diminution of virus-specific Thp in middle aged mice (5 to 11 mo old) could be thought to reflect the time-related loss of viral protein-Ig complexes presented on the surface of FDCs (1). However, experiments with the Ig-/- B6.µMT mice indicate that Ig complexes are not required for either the generation or maintenance of an influenza-specific Thp set (11). It is possible that "low affinity/avidity" cross-reaction (29, 30, 31, 32) with other peptide-MHC class II glycoprotein complexes tends to sustain the memory T cell pool. This is not likely to explain progressive accumulation of virus-specific Thp in the older mice. The long term survival of virus-specific CD4+ T cells would seem to depend more on physiologically determined processes than on the persistence of the inducing Ag (1, 2). In support of this, as has been shown by Tough and Sprent (17), the CD4+CD44high population, which contains the Sendai virus-specific CD4+ memory T cells, is clearly turning over at a fairly constant rate throughout life. This continual turnover, combined with the increased prevalence of CD44highCD4+ T cells in older mice, may be sufficient to explain the accumulation of Sendai-specific Thp.
Neither the increase in virus-specific Thp numbers in very old mice nor the trend for these lymphocytes to be found predominantly in the spleen (after 8 to 12 mo) has been recognized previously for studies of CD8+ T cell memory, although contemporary comparisons of Thp and CTLp frequencies have not been made. What is clear from the current experiments is that the partitioning of the CD4+ Thp set is not readily explained by the staining profiles for CD62L, the lymph node homing receptor (21, 22, 23). A substantial proportion of both the influenza-specific CTLp and Sendai-specific Thp populations revert (10, 33) to the "naive" CD62Lhigh phenotype with time. This should allow these T cells to interact with the appropriate ligands on the high endothelial venules that "gate" lymphocytes into the regional nodes (22). Perhaps this mechanism does operate with the CTLps, but the present results indicate that both CD62Lhigh and CD62Llow Thps tend to be more prevalent in the spleen. One possibility is that memory CD4+ Thps are less motile than the comparable CD8+ set, which could be thought to contain the surveillance T cells that migrate through somatic tissues seeking changes in self-MHC class I glycoproteins (34). Perhaps many of the CD8+ CTLps enter the lymph nodes in the afferent lymph rather than via the blood/high endothelial venule pathway (22, 34).
The increase in prevalence of the Sendai virus-specific Thp population with age is likely to result, at least in part, from the progressive decrease in numbers of naive T cells that follows thymic involution (35, 36, 37). Some of this apparent enrichment could also reflect the return of Thps from the "diaspora" to the spleen. If clonal senescence is a factor for murine (38, 39) CD4+ memory T cells, this does not emerge under the microculture conditions of the LDA assay. Also, the CD4+CD44high T cells seem to proliferate at comparable rates in young and old mice. However, it is important to recognize that there is no information on the relative life spans of the individual lymphocytes within a clone of memory T cells. Accumulation could result from the "experienced" Thps surviving longer. Although the present analysis does provide some insight into the persistence of virus-specific CD4+ T cell memory, many potentially relevant variables remain undefined.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Peter C. Doherty, Department of Immunology, St. Jude Children’s Research Hospital, 332 North Lauderdale, Memphis, TN 38105. E-mail address:
3 Abbreviations used in this paper: LDA, limiting dilution analysis; Thp, T helper cell precursor; B6, C57BL/6J; i.n., intranasal; CLN, cervical lymph node; MLN, mediastinal lymph node; ELISPOT, enzyme-linked immunosorbent spot-forming (assay); BrdUrd, bromodeoxyuridine; CTLp, cytotoxic T lymphocyte precursor; NAPC, normal Ag-presenting cell; VAPC, virus-infected Ag-presenting cell; WBC, white blood cell; EID50, 50% egg infectious dose.
Received for publication December 4, 1997. Accepted for publication June 23, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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