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International Immunology Advance Access originally published online on February 15, 2006
International Immunology 2006 18(4):515-523; doi:10.1093/intimm/dxh392
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© The Japanese Society for Immunology. 2006. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

The phenotype and survival of antigen-stimulated transgenic CD4 T cells in vivo: the influence of persisting antigen

C.-P. Yang1, S. M. Sparshott1, D. Duffy1, P. Garside2 and E. B. Bell1

1 Division of Immunology, Life Sciences Faculty, University of Manchester, UK
2 Division of Immunology, Infection and Immunity, University of Glasgow, UK

Correspondence to: E. B. Bell; E-mail: eric.bell{at}manchester.ac.uk


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
Naive and primed/memory CD4 T cells are distinguished by changes in the expression of activation/adhesion molecules that correspond with an altered function. Adoptively transferred TCR transgenic (tg) CD4 T cells specific for ovalbumin peptide (OVA-pep) were analysed for changing phenotype and the speed of change in vivo following antigen challenge with alum-precipitated (ap) OVA-pep, a conjugate that stimulated a Th2-type cytokine response. The change of CD45RB in relation to number of divisions showed that the transition from CD45RBhi (naive) to CD45RBlow (primed/memory) was incremental; with each cell cycle the number of CD45RBhi molecules on the cell surface was diluted by approximately half and replaced by the low-weight isoform. Similarly, the change to CD44hi expression increased gradually during four rounds of proliferation. The loss of CD62L expression occurred early and was independent of cell division. CD69 was up-regulated quickly within 1–2 cycles, but down-regulated after about seven divisions. The expression of CD49d was not altered during the early rounds of division, although it was up-regulated on 30–60% of tg T cells dividing repeatedly (≥8 cycles). When analysed on day 3 following stimulation, CD25 was no longer up-regulated. The intra-peritoneal injection of ap-OVA-pep stimulated tg T cells in the spleen and mesenteric lymph node one day in advance of those in more distant peripheral lymph nodes. Evidence indicated that residual antigen persisted for at least 4 weeks and was able to stimulate naive tg T cells. However, residual antigen had no net effect on extending or reducing survival of the transferred population.

Keywords: CD45R, CD4 T cells, DO11.10, T cell memory


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
The introduction of antigen either deliberately (by vaccination) or through natural infection initiates a chain of events which culminates in the generation of effector (end-stage) cells, designed to control or eliminate the initial insult, and memory cells responsible for enhancing immunity on subsequent encounter (1, 2). It is possible to reliably identify primed/memory B cells (e.g. by a switch in Ig isotype) (3–5) and primed CD8 T cells, e.g. with the use of peptide-specific MHC class I tetramers (6, 7). However, distinguishing CD4 memory T cells from effector T cells has proved to be more problematic; many of the phenotypic changes which occur after stimulation are reversible, not permanent (8–10). In addition, naive antigen-specific CD4 T cells appear to exist at very low frequencies and do not undergo the extensive proliferation observed with CD8 T cells (6, 11, 12).

High and low molecular weight isoforms of the leukocyte common antigen CD45 have been used extensively to identify naive (CD45Rhigh) and primed/memory (CD45Rlow) CD4 T cells (13). Previous work in the rat, designed to assess CD4 T cell function either by inducing a contact sensitivity response or by providing ‘help’ for memory B cells, showed that surface expression of the CD45Rlow isoform on antigen-primed CD4 T cells was not fixed and with time defaulted back, both phenotypically and functionally, to a resting CD45Rhigh naive phenotype (14–16). A similar reversion was reported by Andersen and Smedegaard (17) when investigating CD4 T cells that protect mice against Mycobacterium tuberculosis. Indirect evidence from human studies also suggested that antigen-primed CD45RO CD4 T cells reverted to a CD45RA naive phenotype (10).

To study antigen-specific CD4 T cells directly, the present investigation used CD4 T cells from DO11.10 transgenic (tg) mice that express a TCR specific for a peptide (pep) of ovalbumin (OVA) (18). Donor tg T cells were transferred into BALB/c recipients and stimulated with OVA-pep, a model developed by Jenkins' group (19, 20). To induce a Th2-type cytokine response, an alum-precipitated (ap) adjuvant was used (21) in place of the frequently used CFA (19, 22). Recipient mice were injected intra-peritoneally (i.p.) instead of the more common subcutaneous route. The intention was to develop a model in which antigen-primed tg T cells could be generated in vivo, recovered and used to explore the basic characteristics of CD4 memory T cells by adoptive transfer.

Here we describe the phenotypic changes which occurred in tg T cells after antigen stimulation; we have monitored the kinetics of up- and down-regulation of activation/adhesion molecules in relation to consecutive rounds of cell division. Our earlier work (14, 15), and that of others (16), suggested that antigen which persists may influence the phenotype of CD4 T cells. Hence, as part of attempts to generate a long-lived tg memory T cell population, we now asked whether residual antigen promoted or inhibited cell survival, induced proliferation or expanded/contracted the size of the antigen-specific memory T cell pool.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Mice
BALB/c (Harlan-Olac, Bicester, UK), BALB/c.Ighb (MK Jenkins, Minneapolis, MN, USA) and DO11.10 mice on a BALB/c background (18) (University of Glasgow) were bred and maintained under barrier conditions in the animal unit, University of Manchester. DO11.10-SCID mice (University of Glasgow) were bred and maintained under SPF conditions in filter cages in Manchester. Mice were used at 6–12 weeks of age.

Antibodies, antigens and reagents
Anti-clonotypic mAb KJ1-26 (23) which recognises the OVA-pep-specific TCR was produced from a hybridoma cell line held by the Scottish Antibody Production Unit (Carluke, Scotland) and purified using Prosep affinity chromatography. KJ1-26 mAb was biotinylated (bio-) with EZ-LinkTM Sulfo-NHS-biotin (Pierce, Rockford, IL, USA) according to the manufacturer's instructions. The following rat anti-mouse mAbs (clone in parentheses) were purchased from BD-Pharmingen (Cowley, UK): CD4, bio-CD4 (GK1.5), PE-CD4 (RM 4-5), FITC-CD45RB, PE-CD45RB (16A), FITC-CD44, PE-CD44 (1M7), FITC-CD62L, PE-CD62L (MEL-14), PE-CD25 (PC 61), PE-CD49d (SG 31), PE-CD154 (MR1), CD19 (1D3), CD8 (53-6.7), B220 (RA3-6B2) and CD16/CD32 ‘Fc block’; hamster anti-mouse PE-CD69 (H1.2F3) and PE-anti-Fas ligand (MFL3) also from BD-Pharmingen. Biotinylated antibodies were detected with streptavidin (SA) Tri-colour (Metachem Diagnostics Ltd, Piddington, UK) or SA-PE (Sigma, Poole, UK). Mice were injected with OVA (Grade V, Sigma) or OVA-pep 323–339 (synthesized by Sigma Genosys, Cambridge, UK) by the i.p. route. Antigen was injected in soluble form (sol-OVA) or after alum precipitation (ap-OVA, ap-OVA-pep). Carboxy fluorescein diacetate succinimidyl ester (CFSE) was purchased from Molecular Probes (Eugene, OR, USA).

Cells
Spleen and the following lymph nodes (LNs), mesenteric LNs (MLNs), inguinal LNs (ILNs), brachial LNs and axillary LNs, were removed, teased apart in PBS containing 2% FCS (PBS–FCS) and filtered through nylon mesh to obtain a single-cell suspension, washed and stained for two- or three-colour flow cytometric analysis. Red blood cells were lysed from spleen cell suspensions by incubating cells in hypotonic Boyles medium at 37°C for 5 min. Cells were washed and resuspended in PBS–FCS. A total of 0.5–1.0 x 106 cells were stained for FACS analysis; non-specific binding was inhibited by the addition of 1 µl of Fc block. Live lymphocytes were gated electronically and 104 to 3 x 105 cells acquired for analysis. T cells used for adoptive transfer were obtained from LNs (not spleen) of DO11.10 donors and purified for CD4 T cells by depleting the population of CD8 T cells and B cells. Cells were stained with rat anti-mouse mAb against CD8, CD19 and B220, washed, mixed with anti-rat Ig conjugated Biomag® particles (Metachem Diagnostics) and the cells adhering to the ferric particles removed by applying a strong magnet to the side of the tube. The resulting population was >95% CD4+ of which 70–80% were KJ1-26+ (abbreviated KJ+ T cells). LN cells from DO11.10-SCID donors were injected without purification and were 95–97% CD4+ KJ1-26+. The procedure for labelling LN cells with CFSE was modified from Lyons and Parish (24). A frozen aliquot of 5 mM CFSE in dimethyl sulphoxide was diluted 1/50 or 1/100 in PBS; 10 µl of dilute CFSE was added to 1 ml of 107 cells ml–1 in 37°C PBS, incubated for 10 min at 37°C and the reaction stopped by adding 100 µl FCS per 1 ml of cells. The cells were washed twice in cold PBS–FCS and resuspended at the appropriate concentration for injection, 0.2 ml intravenously (i.v.)


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Kinetics of response
As part of an investigation using tg CD4 T cells to provide help for memory B cells (D Duffy, C-P Yang, A Heath, P Garside, EB Bell, submitted for publication), BALB/c recipients were injected i.v. with CFSE-labelled CD4 T cells from DO11.10-SCID donors and challenged i.p. with ap-OVA-pep, an adjuvant conjugate that induces a Th2-type cytokine response (21). Analysis of the MLN for the presence of CD4 tg T cells showed that their numbers increased from day 2, reached a maximum by day 7 and then declined (Fig. 1). The splenic response was identical (data not shown). The KJ+ T cell response in the ILN, distant to the site of injection, started a day later.


Figure 1
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  		Fig. 1. Kinetics of clonal expansion induced by the i.p. injection of alum-precipitated OVA-pep. CFSE-labelled tg CD4 T cells (3 x 106) from LNs of DO11.10-SCID donors were injected i.v. and BALB/c recipients challenged (solid symbols) or not (open symbols, No Ag) the next day with 100 µg ap-OVA-pep. Recipients were killed at daily intervals and the MLN and ILN analysed for the presence of CD4+ KJ1-26+ (KJ+) T cells. The results from four independent experiments were pooled. Each point represents the mean ± the range of 2 or ± SD of 4 recipients.

 
Phenotypic changes in vivo
Antigen-stimulated CD4 T cells undergo a number of phenotypic changes including the expression of activation and adhesion molecules that, in turn, have been used to identify ‘primed’ and/or ‘memory’ T cells. In the mouse different isoforms of CD45RB have been used to distinguish naive (CD45RBhi) from antigen-primed (CD45RBlow) CD4 T cells (25). To investigate the transition from CD45RBhi to CD45RBlow expression, KJ+ T cells obtained from LNs of DO11.10-SCID donors were labelled with CFSE, injected into BALB/c recipients (Fig. 2A) and stimulated the following day by i.p. injection of 100 µg ap-OVA-pep. Quantitative decreases in levels of cytoplasmic CFSE (Fig. 2B) allowed the subsequent detection of seven defined cycles of cell division in mice receiving antigen (Fig. 2E). In the absence of antigen the KJ+ T cells did not divide significantly (Fig. 2C). To analyse CD45RB expression, the KJ+ population was partitioned on each day into increments (Regions 1–9) defined by CFSE fluorescence (Fig. 2E; R1 = no cell division; R9 = >8 cycles of division). Each gated region was analysed for CD45RBhi/RBlow staining (Fig. 2F). It was noted that the intensity of CD45RB staining declined after each cell cycle in a stepwise fashion with the result that the change from CD45RBhi to RBlow was gradual (Fig. 2D). An analysis of CD45RB showed that 2–3 cycles of division were required before appreciable numbers of CD45RBlow KJ+ T cells were observed (Fig. 3A). By 5–6 days after challenge the entire cohort of donor cells was recruited into the response, all had divided at least once (Fig. 3B) and nearly all KJ+ T cells expressed the primed CD45RBlow phenotype (Fig. 3C). KJ+ T cells that were not stimulated maintained a CD45RBhi phenotype [Fig. 3C; no antigen (No Ag)]. By 10 days, more than 80% of donor cells had undergone more than 8 cycles of division (Fig. 3B). There were significant differences between the responses in the MLN (and spleen, data not shown) and those in the ILN. The reduction in CD45RB expression was first observed in the MLN at day 2, a day before a similar reduction was detected in the more distant ILN (Fig. 3C), a change that coincided with the start of cell division in these tissues.


Figure 2
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  		Fig. 2. Analysis of CD45RB expression in relation to number of cycles of division. Recipients for the analysis were from those in Fig. 1. MLN and ILN cells were stained with KJ1-26 plus anti-CD45RB or anti-CD4 mAbs and analysed for CFSE content by three-colour flow cytometry. Results were pooled from four independent experiments. (A) Representative profile showing the donor KJ+ T cells (R1) from the MLN 3 days after ap-OVA-pep challenge. (B) A profile of the R1-gated population shows the quantitative reduction of CFSE expression among the dividing KJ+ T cells. (C) In the absence of antigen challenge the KJ+ T cell population failed to divide and retained the original level of CFSE staining. The profile shows the MLN 6 days after transfer (No Ag). (D) A representative profile shows the progressive reduction (linear regression) in CD45RB expression with diminishing levels of CFSE. (E) The gated KJ+ T cell population (day 3 MLN) was divided into regions (R1–R9) on the basis of diminishing levels of CFSE in which R1 represents no division, R2 one cell division, etc. and R9 >8 cycles of division (right to left). (F) The content of each region was analysed with respect to CD45RB expression to determine the percentage of CD45RBhi and CD45RBlow KJ+ T cells within each cycle.

 

Figure 3
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  		Fig. 3. The change from CD454RBhi to CD45RBlow occurred in a stepwise sequence (A) and was linked with each cycle of division. (B) The KJ+ T cell population, recovered from the MLN at selected days, was analysed with respect to number of cell cycles. Points represent the proportion of the total KJ+ T cells (100%) that did not divide (0 divisions) or divided 1 to >8 times, right to left. In the absence of antigen (No Ag), >90% of the KJ+ population failed to divide even once. (C) Comparison of changes in CD45RB expression of KJ+ T cells in the MLN versus the ILN following challenge (solid symbols). In the absence of antigen (open symbols), KJ+ T cells remain CD45RBhi. Each point in A, B and C represents the mean of 2–6 recipients.

 
A similar strategy was adopted to investigate expression of the following activation and adhesion molecules in response to antigen challenge in vivo: CD44, CD62L, CD69, CD49d, CD25, CD40L and CD95. The analysis was undertaken on day 3 after antigen challenge, a time when the majority of KJ+ T cells was starting to divide, day 7 at the peak of the response and at day 10 when KJ+ T cell numbers were in decline (data not shown). CD44, a marker often used to identify memory T cells (25), was up-regulated following cell division and by day 7 nearly all KJ+ T cells were CD44hi (Fig. 4a). KJ+ T cells that failed to divide retained their CD44low phenotype (Fig. 4a, events on the far right of profile). The expression of CD44 started to increase following 1 or 2 cell cycles (Fig. 4b) and by 3–4 divisions reached maximum levels. CD62L (L-selectin), an adhesion molecule that interacts with GlyCAM-1, is expressed on LN high-walled endothelial venules (HEVs) and facilitates the first stage in migration of lymphocytes from blood into LNs (26, 27), was shed rapidly (Fig. 4c and d). Whereas ~90% of KJ+ T cells expressed CD62L before transfer (Fig. 4d, Pre), <50% of KJ+ T cells remained CD62L+ by the start of proliferation. With time, a fraction of the antigen-stimulated KJ+ T cells re-expressed CD62L (Fig. 4d, day 7, >8 cycles of division). The T cell activation molecule CD69 was up-regulated on 50–70% of KJ+ T cells before cell division and was maximum (70–80% of dividing cells) after 2–3 cell cycles (Fig. 4e and f). Subsequently, CD69 expression declined as KJ+ T cells underwent further rounds of proliferation.


Figure 4
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  		Fig. 4. Analysis of activation and adhesion molecule expression in relation to cycles of antigen-induced division by KJ+ T cells in vivo. KJ+ T cells from D011.10-SCID LNs (a–d) or CD4 T cells from DO11.10 LNs (e–j) were labelled with CFSE and transferred to BALB/c mice. The following day (day 0) recipients were injected i.p. with 100 µg ap-OVA-pep, killed on day 3 (open bars) or day 7 (filled bars), MLN and axillary LNs were pooled and stained with KJ1-26 together with mAbs against the following surface molecules: CD44 (a and b), CD62L (c and d), CD69 (e and f), CD49d (g and h) or CD25 (i and j). Representative profiles of gated donor cells on day 7 are illustrated for MLN only (c and d) or MLN and axillary LNs pooled (e, g and i). The latter three profiles include wild-type KJ– CFSE+, non-dividing donor cells on the far right. The KJ+ T cell population was gated to exclude the KJ– donor cells, partitioned into regions on the basis of diminishing levels of CFSE as illustrated in Fig. 2D, and each region analysed with respect to activation and adhesion molecule expression (b, d, f, h and j). Each histogram within b, d, f, h and j represents the percentage of KJ+ T cells expressing the molecule in question before (0 divisions) or after 1 or more (1 to >8) cycles of division. The phenotype of the KJ+ T cell population before injection (Pre) is illustrated for CD44 and CD62L (b and d). The results were pooled from three independent experiments. Values are means ± SD of 4 recipients per group.

 
CD49d represents the {alpha}4 subunit of the integrins {alpha}4ß1 and {alpha}4ß7 expressed at low levels on naive CD4 T cells and at higher levels on a fraction of the CD45RBlow CD44hi subset (27–29). Pooled mesenteric and axillary LNs from KJ+ T cell-injected recipients were assessed with regard to the percentage of CD49d+ donor cells (Fig. 4g and h). In addition, the median fluorescence intensity (MFI) within the CD49d+ population was recorded as a measure of the density of {alpha}4 expression on the surface (Table 1). Following antigen stimulation the percentage of KJ+ T cells expressing CD49d increased but only on those tg T cells that had undergone more than 5–6 cycles of division (Fig. 4h). Not only did the number of CD49d+ T cells increase but the MFI was also significantly higher compared with wild-type KJ– CD4 T cells transferred from the DO11.10 strain (Table 1); note that the latter did not divide. When KJ+ T cells were gated on those undergoing 1–6 cycles or ≥8 cycles of division, it was apparent that only after repeated division did the concentration of CD49d significantly increase (Fig. 4g; Table 1, Experiment 1). Transferring KJ+ T cells from DO11.10-SCID donors produced a similar result (Table 1, Experiment 2). In this experiment the 10-fold reduced CFSE content precluded a detailed analysis beyond 4 cycles.


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  		Table 1. The intensity of CD49d expression on KJ+ T cells increases after 4–6 cycles of division

 
Changes in CD25 expression (the IL-2{alpha} receptor) were minimal on days 3 and 7 (Fig. 4i and j) except among KJ+ T cells that had divided repeatedly (>8 cycles) and this was only apparent early in the response (day 3). By 7 days, nearly all remaining KJ+ T cells had been stimulated into multiple rounds of proliferation, but very few (<7%) were CD25+. We were unable to detect any staining for CD40L (CD154) or CD95 (FasL) on KJ+ T cells activated in vivo.

Effect of antigen dose on antigen-primed KJ+ T cells
Naive KJ+ T cells in normal recipients, stimulated the following day with antigen, underwent repeated cycles of cell division. The number of KJ+ T cells increased and then rapidly contracted (Fig. 1). By day 28 the number of KJ+ T cells remaining was below the starting frequency (D Duffy, C-P Yang, A Heath, P Garside, EB Bell, submitted for publication). To ensure that the dose of antigen used to challenge recipients was not contributing to the early demise of the expanded population, recipients of KJ+ T cells were injected with 10 or 1 µg ap-OVA-pep, i.e. 10-fold and 100-fold lower than the standard dose. The effect of reducing the dose 100-fold was to reduce the degree of proliferation (Fig. 5, day 7). The number of KJ+ T cells surviving by day 14 was not altered by the lower doses of antigen.


Figure 5
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  		Fig. 5. Low antigen dose did not prevent contraction of the KJ+ T cell population. 4–6 x 106 CD4 T cells, purified from LNs of D011.10 donors and CFSE labelled were injected i.v. to two groups of BALB/c recipients. The following day (day 0) recipients were injected i.p. with 10 or 1 µg ap-OVA-pep and killed on days 7 and 14. MLNs were analysed for the presence of CD4+ KJ+ T cells. Histograms represent means ± SD of 3 recipients per group.

 
Persisting antigen affects phenotype but not survival
Although it is possible that antigen remaining after the initial injection may continue to exert an effect on specific T cells, few studies have investigated this issue systematically. To determine what influence persisting antigen could have on naive KJ+ T cells, recipient mice were injected with 100 µg ap-OVA in advance (–2 months, –1 month and –2 weeks), i.e. before the transfer of KJ+ T cells on day 0. No antigen was administered after cell transfer. Recipients were killed at intervals (1, 7 and 14 days) and MLN, ILN and spleen analysed by flow cytometry for the presence of donor T cells and CD45RB expression. The changes in the percentage of KJ+ T cells were too small (data not shown) to determine with any certainty whether or not the KJ+ T cell population was responding to persisting antigen. However, there were significant changes in CD45RB expression in all tissues examined, linked with the presence of residual antigen. In the absence of OVA (No Ag), the KJ+ T cell population remained unchanged, 91–95% CD45RBhi (Fig. 6A–C). In recipients where ap-OVA had been injected 2 weeks prior to cell transfer, the percentage of KJ+ T cells expressing the naive phenotype (CD45RBhi) had decreased significantly by day 14 in all tissues, for example to 55% in the MLN (Fig. 6A). Significant reductions in CD45RBhi expression were also recorded in MLN, ILN and spleen from recipients injected 1 month in advance of KJ+ T cell transfer. By 2 months following injection, persisting antigen had largely disappeared as there was no evidence of a significant population of CD454RBlow KJ+ T cells. Given that the change from CD45RBhi to CD45RBlow required several rounds of antigen-induced cell division (Figs 2 and 3), the loss of CD45RB expression appeared to be a sensitive measure for monitoring the presence or absence of residual antigen.


Figure 6
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  		Fig. 6. Antigen that persists in vivo following immunization activates naive KJ+ T cells as determined by changes in CD45RBhi expression. Groups of BALB/c recipient mice were injected i.p. with 100 µg ap-OVA-pep 2 weeks (–2 weeks), 1 month (–1 month), 2 months (–2 month) or left uninjected (No Ag) before receiving (day 0) 5 x 106 CD4+ KJ+ T cells (i.v.) from LNs of D011.10 donors. Recipients were killed on days 1, 7 and 14, MLN (A), ILN (B) and spleen (C) stained with KJ1-26, anti-CD4 and anti-CD45RB mAbs and analysed by flow cytometry. CD45RBhi expression was assessed on KJ+ T cells. The results of two independent experiments were pooled. Each value represents the mean ± SD of 3–6 recipients, except for a single recipient analysed on day 1 of the –1 mo group. Changes in CD45RBhi expression: –2 weeks and –1 month versus No Ag; *P < 0.05, **P < 0.02, ***P < 0.01.

 
The experiments above indicated that naive CD4 T cells could acquire an effector/memory phenotype (CD45RBlow). Therefore, we asked whether the lifespan of KJ+ T cells could also be affected by persisting antigen. Groups of BALB/c mice were uninjected (No Ag) or injected with 100 µg ap-OVA 2 weeks or 1 month before (–2 weeks, –1 month) the transfer of purified CFSE-labelled KJ+ T cells. Recipients were killed at weekly intervals and analysed by flow cytometry for the presence of donor cells. In all three groups, donor cells declined steadily (apart from a possible increase on day 7 within the –2 week group), were almost undetectable (<0.01%) by day 21 and had virtually disappeared by day 28 (Fig. 7). The presence of persisting antigen made no discernible impact on KJ+ T cell survival. The experiment was repeated with essentially the same result.


Figure 7
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  		Fig. 7. Persisting antigen does not influence the survival of naive KJ+ T cells. Groups of BALB/c mice were uninjected (No Ag) or injected i.p. with ap-OVA-pep 2 weeks (–2 weeks) or 1 month (–1 month) in advance of cell transfer. All mice were injected i.v. on day 0 with 4 x 106 CFSE-labelled CD4+ T cells purified from LN cells of D011.10 donors and were killed at weekly intervals. MLN cells of recipients were stained with KJ1-26 and anti-CD4 mAbs and analysed by flow cytometry for the presence of donor CD4+ KJ+ T cells. The results of the two experiments were pooled. Values represent means and range of 2 recipients per group.

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
The present study set out to explore the changes that occurred in tg CD4 T cells in response to immunization with a Th2-type adjuvant. We have shown elsewhere (D Duffy, C-P Yang, A Heath, P Garside, EB Bell, submitted for publication) that KJ+ T cells transferred together with naive B cells to SCID recipients and immunized with ap-OVA-pep generated predominantly a Th2-type IgG1 class of antibody. Most published work on DO11.10 T cells has been based on Th1 adjuvants, CFA or LPS (20, 22, 29, 30). For the present investigation donor tg T cells were obtained from the DO11.10-SCID strain, thus avoiding the transfer of cells contaminated with wild-type or KJ+ T cells expressing both tg and endogenous TCRs (31). Instead of injecting OVA into a subcutaneous site which concentrates the response in draining LNs, antigen was injected i.p. The peak of the KJ+ T cell response occurred at day 7, two days later than that recorded using Th1-type adjuvants (19, 20). Interestingly, KJ+ T cells in the MLN and spleen were the first to respond, a day in advance of KJ+ T cells in more distant sites including the ILN (Fig. 3C), axillary and brachial LNs (data not shown). The delay in the peak response could be a function of the alum and the site of injection (i.p.). A restricted release would delay antigen reaching distant LNs and tend to prolong the time before the response reached maximum. The early response in MLN and spleen indicated by proliferation (CFSE) and a switch from CD45RBhi to CD45RBlow suggested that OVA was delivered directly to these tissues from the peritoneal cavity, although the route by which this occurred was not clear; lymphatics drain cranially from the peritoneal cavity to the mediastinal LNs (32).

CD4 T cells that encounter specific antigens undergo a series of membrane alterations that distinguish the primed from the naive subset. To determine the transition point at which surface molecules were up- or down-regulated, we analysed the phenotype of CFSE-labelled KJ+ T cells after each cycle of division. We were particularly interested in the kinetics of CD45RB expression and therefore analysed recipients killed on days 1–10 (Figs 2 and 3). Cell division was first observed in the MLN on days 2 and 3 in the distant ILN where a majority of the KJ+ T cells divided 1–4 times. A day later, four additional cycles of division were apparent, suggesting a relatively short (~6 h) cell cycle time. Others have reported similar rapid kinetics in LNs draining a subcutaneous site following the use of a Th1 adjuvant (19, 30). We found that by day 10 ~70% of KJ+ T cells had divided more than eight times. The number of CD4 T cells recruited into the response was determined by antigen dose and the number of KJ+ T cells transferred. Injecting 100 µg ap-OVA was sufficient to stimulate, by day 3, the entire cohort to divide at least once (Figs 2 and 3). However, reducing the dose of antigen limited the degree of proliferation (Fig. 5) and left many KJ+ T cells undivided (C-P Yang, EB Bell, unpublished observations). Others showed that increasing the transferred KJ+ T cells 25-fold not only reduced the number of cell cycles overall, but also resulted in many KJ+ T cells failing to divide even once by day 3 (33). These observations probably reflect competition by antigen-specific T cells for a limited resource—antigen.

The change in CD45RB expression among individual T cells was not immediate, but gradual. After the first and each subsequent cell cycle the CD45RBlow phenotype of the dividing population increased in a stepwise fashion, conforming to the pattern of a linear regression (Fig. 2D). The entire cohort of injected cells did not loose CD45RBhi expression completely until days 5 or 6. When T cells from DO11.10 mice were stimulated with OVA-pep in vitro, a similar observation was reported, i.e. a gradual loss of CD45RBhi (34). Changes in membrane expression of CD45R occur by the synthesis of new molecules generated by the differential splicing of the variable exons 4, 5 and 6 encoding, respectively, the restricted epitopes CD45RA, B and C (35). Given that membrane density of CD45R is relatively constant (36), the present observations suggest that after each division the cell membrane of the two new daughter cells may be a composite of the older CD45R isoforms diluted by newly synthesized low molecular weight isoforms called CD45RBlow in mice, CD45RC– in rats and CD45RO in humans. The gradual replacement of CD45Rhi with CD45Rlow isoforms [and a replacement in the reverse direction when primed CD4 T cells revert to a resting state (14, 15, 37)], accounts for the lack of a clear division between the two subsets when analysed by flow cytometry.

The tyrosine phosphatase activity of the cytoplasmic tail of CD45 represents a brake, inhibiting lck-induced T cell activation (38). During TCR-induced stimulation, CD45 is mobilized to the periphery of the immune synapse (39, 40). It was suggested that the smaller CD45Rlow molecule allowed faster exclusion of the CD45 phosphatase inhibitor from the synapse, enabling an accelerated response to take place (41). As the number of CD45Rhi molecules decreases with each cell division, the time between cell cycles would shorten and could account for the relatively short cell cycle times observed between days 2 and 3.

The up-regulation of CD44 has been used widely to identify primed/memory T cells in mice (1, 2). CD44 has certain similarities to CD45. Both molecules are encoded by single genes with multiple exons which can be differentially spliced (35, 42). Unlike CD45 which has no known ligand (36), CD44 binds to hyaluronic acid (43), a component of the extracelluar matrix; this pairing promotes extravasation into sites of inflammation (43, 44). The change of this activation marker from CD44low to CD44hi occurred with similar kinetics (Fig. 4a and b), although in the opposite direction to that observed for CD45RB. There was a distinct stepwise up-regulation of CD44 during the first 3–4 cycles of division. The very few KJ+ T cells that failed to divide by day 7 retained their CD44low phenotype. Interestingly, when examined many weeks after immunization, KJ+ T cells were found to have reverted to CD44low but to have retained the CD45RBlow molecule (30).

CD45 and CD44 appear to work in concert. The phosphatase activity of the CD45Rhi isoform ensures the T cell remains in a quiescent state until TCR engagement. Whereas highly gylcosylated CD45Rhi molecules are excluded from the immune synapse (39, 40), the CD45Rlow isoform may drift back into the raft (45), perhaps to prevent the immediate production of cytokines within lymphoid tissue. On the up-regulation of adhesion molecules and chemokine receptors, the T cell acquires the ability to migrate into inflammatory sites where the up-regulated CD44 can now engage hyaluronic acid initiating local cytokine ({gamma}-IFN) production (46).

The lymphocyte ‘homing receptor’, CD62L, enables lymphocytes to enter LNs from the blood across HEVs by binding to its endothelial ligand GlyCAM-1 (26). CD62L is shed rapidly from the membrane during activation but is re-expressed on memory CD4 T cells that return to a resting state (47). The present in vivo studies showed that the early loss of CD62L was not linked with cell division, confirming a similar conclusion from work in vitro (48). The shedding of CD62L prevents tethering to HEVs and deprives the cell of its ability to re-enter peripheral LNs. At the same time, T cells acquire adhesion molecules including the CD49d+ integrins {alpha}4ß7 and {alpha}4ß1 which enables them to infiltrate non-lymphoid tissues.

The {alpha}4 subunit, CD49d is expressed at low levels on naive CD4 T cells in the MLN (27, 28) and on a few activated T cells in peripheral LNs (26). {alpha}4ß7 is up-regulated on a fraction of the antigen-stimulated CD45RBlow CD44hi (mouse) or CD45RA– (human) CD4 T cells (29, 49) and promotes selective migration into mucosal lymphoid tissues—Peyer's patches and gut lamina propria (26, 27). Expression of the {alpha}4ß1 integrin enables migration across vascular endothelium at sites of inflammation (26). The present analysis reflects {alpha}4ß7 expression in the MLN; the inclusion of the axillary LN (~10% of the total) would contribute marginally to the CD49d staining. Changes in CD49d on KJ+ T cells have not been studied apart from the interesting observations by Campbell and Butcher (29) using soluble OVA, injected i.p. with the Th1 stimulant LPS. They showed that {alpha}4ß7 was up-regulated by day 2 on 30–60% of KJ+ T cells in the MLN but not in peripheral LNs (inguinal, brachial). In contrast, tg T cells in peripheral LNs expressed the ligand for P-selectin (P-lig). There were two distinct differences observed from the present study that used a Th2 adjuvant. Following OVA/LPS, (i) the proliferative burst was well advanced by day 2 in both MLN and peripheral LNs with a larger majority of the KJ+ T cells stimulated into division; (ii) {alpha}4ß7 was up-regulated quickly in a stepwise fashion starting after a single-cell division. Using the Th2 antigen, by day 2 the proliferative response was only just starting in the MLN. Secondly, there was no evidence that {alpha}4 was up-regulated until KJ+ T cells had undergone more that 6 cycles of division (Table 1, Fig. 4). Among those tg T cells that had divided extensively, 30–60% expressed the {alpha}4 subunit when examined at days 3 or 7. In contrast, using the Th1 adjuvant 30–60% of the entire tg cohort expressed {alpha}4ß7 by day 2 (29); the comparable fraction in the present study was 12.7% ± 4.4 at day 3. Apparently, few KJ+ T cells were primed for migration to mucosal sites by the Th2 adjuvant.

CD25, an early activation molecule, was found on large numbers of OVA-stimulated KJ+ T cells within 6 h of injection with CFA (30). By 24 h, 90% of tg T cells expressed high levels of the IL-2R. However, 2 days later, CD25 had returned to baseline values. Similarly, by day 3 we observed CD25 only on a small fraction of tg T cells and expression was not a function of cell division.

The extent to which residual antigen may modify the fate of CD4 memory T cells (death or survival) has yet to be resolved. The work of previous investigators suggested that persisting antigen could affect the phenotype and function of CD4 memory T cells, for example by preventing CD45Rlow CD4 T cells from reverting to a CD45Rhi naive phenotype (14–16, 37). Using naive tg T cells we found that persisting antigen did not stimulate sufficient proliferation to register as an increase in the number of KJ+ T cells. Nevertheless, there was clear evidence of an altered phenotype; the percentage of tg T cells expressing the primed CD45RBlow isoform increased significantly in recipients injected with ap-OVA-pep 2 weeks and 1 month before cell transfer. In the complete absence of antigen, KJ+ T cells fail to divide or to become activated (Fig. 2) (19, 20, 37). In the present study the non-dividing T cells remained entirely CD45RBhi (Fig. 3) and the switch to CD45RBlow only occurred among KJ+ T cells that had undergone several rounds of proliferation. The reduction in the percentage of CD45RBhi KJ+ T cells was used as a surrogate marker of antigen-induced stimulation. In a recent study naive tg CD4 T cells specific for an epitope of the influenza virus were found to proliferate, down-regulate CD62L and become committed to {gamma}-IFN production by persisting residues of antigen (50).

There is no clear evidence to indicate what effect a depot of persisting antigen would have on the size of the memory T cell population. Investigations in humans and rodents monitoring the turnover and lifespan of CD4 T cells showed, unexpectedly, that T cells with a memory phenotype (CD45Rlow) turned over rapidly and had a short lifespan (51, 52). Naive CD4 T cells (CD45Rhi) were slow to divide (in humans approximately once a year) and were long-lived. The implication is that long-term survival of memory populations would depend on periodic low-grade proliferation driven by homoeostatic mechanisms or by occasional encounters with antigen. Recently evidence was published to suggest that retention of viral antigens could recruit naive CD4 T cells into the effector/memory subset (50). Further evidence indicated that residual antigen could have a detrimental effect on the memory pool. Tg TCR CD4 T cells repeatedly exposed in vivo to viral antigen-pulsed dendritic cells were significantly reduced in number and were impaired in function (53). We wanted to determine whether residual antigen could alter the fate of the KJ+ T cell population on transfer either by prolonging their survival or alternatively accelerating their demise. The rapid and extensive contraction of the KJ+ T cell population following the antigen-induced expansion phase is well documented. The transfer of naive KJ+ T cells into recipients injected with ap-OVA-pep 2 weeks or 1 month earlier had no net effect on survival; antigen neither accelerated nor slowed the disappearance of the tg T cells. There was no evidence that residual antigen made a contribution to or compromised the lifespan of the KJ+ T cell population. We have argued elsewhere (13) that residual antigen may play a role in maintaining some T cells in an up-regulated state, but that long-term memory is based on those CD4 T cells that return to a quiescent state, revert to a naive phenotype and adopt the characteristics of a long-lived naive CD4 T cell. Memory in these circumstances would depend on the increase in frequency, an issue that has yet to be resolved.


   Acknowledgements
 
This work was supported by grants from the British Biotechnology and Biological Sciences Research Council (to E.B.B. and P.G.) and the Arthritis Research Council, UK (E.B.B.).


   Abbreviations
 
ap   alum precipitated
CFSE   carboxy fluorescein diacetate succinimidyl ester
HEV   high-walled endothelial venule
ILN   inguinal lymph node
i.p.   intra-peritoneal
i.v.   intravenous
KJ+ T cell   mAb KJ1-26+ CD4 T cell
LN   lymph node
MFI   median fluorescence intensity
MLN   mesenteric lymph node
No Ag   no antigen
OVA   ovalbumin
pep   peptide
tg   transgenic

   Notes
 
Transmitting editor: T. Hunig

Received 13 July 2005, accepted 2 January 2006.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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