III.5 Mercados potenciales
III.5.2 Patrón de importaciones
M aturation of DCs encom passes phenotypic changes, e.g. upregulation of costimulatory m olecules such B7-2 and CD40, as w ell as functional alterations. To study whether the regulation of costimulation is reflected in the stimulatory capacity of DCs, 1 set out on a series of experiments using FI animals heterozygous for the H-2 region. One of their I-A alleles was compatible w ith a first set of CD4^ T cells which fed back to the DCs (=> "modulators"), while the other allel was the restriction element of the second set of CD4^ T cells ("responders"). Preliminary experim ents were performed to establish that the T cells used in these assays (D O ll.lO and 3A9-RAG2''' ) are not alloresponsive to 1-A'^ or I-A^, respectively, to ensure that any response 1 observe is antigen-specific. Neither D O ll.lO nor 3A9-RAG2''^' CD4^ T cells^ responded to unpulsed DCs from Fl(BALB/c x BIO.BR), BIO.BR or BALB/c mice. Furthermore, D O ll.lO CD4^ T cells only responded to OVA3 2 3 . 3 3 9 peptide presented by BALB/c cells but not by BIO.BR cells, w hile 3A9 CD4^ T cells only responded to HEL4 6 . 6 1 peptide presended by BIO.BR but not by BALB/c cells (data not shown).
N one of the in vitro assays to measure the effect of T-cell feedback on the stimulatory capacity of DCs yielded conclusive data. Therefore, I tried to address the m odulation of stimulatory capacity of DCs by activated T cells in vivo. Purified CD4^ T cells from D O ll.lO donors were adoptively transferred into FI (BALB/c x BIO.BR) hosts. At the same time, a small number of purified, CFSE-labelled 3A9 T cells were injected into the same hosts. As measured by flow cytometry, each division of CFSE- labeled cells becomes apparent as a 2-fold reduction in mean fluorescence intensity (MFI) in the daughter cells. The day after transfer of the two T-cell populations, the mice were immunised as usual with PBS, OVA3 2 3.3 3 9, CpG, or OVA3 2 3 . 3 3 9 and CpG, and
in addition w ith a dose titration of an agonistic peptide for 3A9 T cells when presented on I-A^. The 3A9 T cells can be identified by staining w ith the anti- clonotypic antibody 1G12. FACS analysis of the CFSE profiles three days after immunisation of the hosts showed clear evidence for proliferation of 3A9 T cells in the spleen at lower doses of (Fig. 4.8 A, arrows second row) w hen D O ll.lO T cells were activated by OVA3 2 3.3 3 9. Moreover, although the repercussion of D O ll.lO T-cell activation on the proliferation of 3A9 T cells was not as pronounced as the effect of CpG injection (third row), it w as readily detectable over the w hole dose range of HEL4 6 . 6 1 tested here. Importantly, activation of D O ll.lO T cells can exert its effect on DCs in the absence of microbial stimulation. These findings are in accord w ith the dem onstration of functional cooperation betw een two populations of naïve CD4^ T cells stim ulated by the sam e AFC, rendering otherw ise silent T-cell epitopes imm unogenic (481). Combination of OVA3 2 3 . 3 3 9 and CpG was most potent in enhancing 3A9 T-cell proliferation. The synergy betw een the two stimuli is m ost effective at low HEL4 6 . 6 1 doses (arrow fourth row). Notably, at the highest antigen dose 3A9 T cells in m ice w hose treatment did not include CpG w ent through m any rounds of division (first and second row, fourth column) without generating a large pool size (data not shown). Their CFSE profile indicates that the transferred cells proliferated rapidly in a single burst (and then possibly died), w hile the corresponding cells in mice treated w ith CpG appear to cycle less rapidly and less synchronous (third and fourth row, fourth column). This is consistent w ith reports that T cell stim ulated in vivo in the absence of endotoxin initially proliferate vigorously and then rapidly die (234). It is important to take into consideration that this is only indirect evidence for signals from D O ll.lO T cells to APCs to increase their potency to activate naïve T cells. It could well be that the D O ll.lO T cells support the 3A9 T cells directly for example by secretion of IL-2, instead of providing help via feedback activation of the AFC, as it came apparent in the experiment shown in figure 4.5 B. This is one of the major disadvantages of this
in vivo system. Here, the conditioning phase and the readout phase ("assaying") are
synchronous and identical, and therefore the m odulating T cells (here D O ll.lO T cells)
are present during the entire experiment. In contrast, in the in vitro experiments the conditioning and assaying of the DCs was done sequentially. This also provides an opportunity to arrest the conditioned DCs in their current status by chemical fixation (see Supplementary Data).
Figure 4.8 Activated CD4^ T cells increase the stimulatory capacity of APCs in vivo
A. Purified DO 11.10 T cells and low num bers of A P C -depleted, C FS E -labelled T cells
purified from 3A9 mice w ere adoptively transferred into F1 (BALB/c x B10.BR) mice. The
follow ing day, mice w ere injected with O VA3 2 3 . 3 3 9 and/or CpG (row labels), in com bination
with the indicated doses of HEL^g-si (column labels). T hree days later, proliferation of 3A9 cells was assessed by FACS analysis of splenocytes. C F S E -histogram s of 3A9 cells are shown (gated on CD4^1G12‘"V(38 TCR+ cells with the appropriate sca tte r profile). Results
are representative of three independent experim ents. B. Purified OT-I I T cells and low
num bers of C F S E -labelled OT-I T cells w ere a doptively transferred into naïve C57BL/6 m ice. The follow ing day, m ice w ere injected w ith o r w ith o u t O V A3 2 3 . 3 3 9 (row labels), in
co m b in a tio n w ith the in d ica te d doses of pO V A (co lu m n la b els). T h re e days later, proliferation of OT-I T cells was assessed by FACS analysis of splenocytes. CFSE vs Va2 TC R density plots are show n (gated on CD4'8^ cells w ith the appropriate scatter profile). Results are representative of three independent experim ents.
0 n E 3 1 10 1 0 0 1000 10000 1 10 1 0 0 1000 10000 1 10 1 0 0 1000 10000 1 1 0 1 0 0 1000 10000 %m so < >
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Figure 4.8 A (figure legend on previous page)
CFSE
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SProvision of help by CD4^ T cells is thought to be important for the activation of naïve CTL precursors. To test whether cognate feedback from new ly activated naive CD4^ T cells increases capacity of APCs to stimulate of CD8^ T cells w ithout the need for a microbial stimulus, C57BL/6 host mice were grafted w ith purified CD4^ cells from OT-II donor mice and purified CFSE-labelled T cells from OT-I donors. Similar to the previous experiment, the hosts were imm unised the next day w ith PBS or OVA3 2 3.3 3 9, in combination w ith a dose titration of pOVA^. Three days later, the CFSE profiles of CD4'CD8^ Va2 TCR^ cells in the spleen was analysed by FACS. Activation of OT-II cells by co-administration of OVA3 2 3 . 3 3 9 augmented the antigen-specific proliferation of OT-I cells markedly over the whole pOVA dose range tested here (Fig. 4.8 B). There is a dim inutive population of Va2 TCR+ cells which have apparently run through one round of cell division in host mice which did not receive any pOVA. Subsequent experiments show ed that this is an artefact, presumably representing dead or dying cells (data not show n). The large population of CFSE V a2 TCR^ cells consists of endogenous CD8^ T cells from the normal C57BL/6 repertoire as w ell as a few CD8^ T cells bearing the OT-II TCR. The OT-II mice used for this study were not bred on a RAG or scid background. FACS analysis of cells from secondary lym phoid organs from OT-II mice revealed that a significant fraction of Va2V|35 TCR-bearing cells are CD8^ and not CD4^ (data not shown).
^ OT-I and OT-II T cells both bear a Va2VpS TCR, and are CD8^ and CD4^, respectively. D O ll.lO and OT-II T cells share the amino acids 323-339 of ovalbum in as an agonistic peptide, and are restricted by I-A‘^ and I-A^ respectively.
no pOVA
i#
0.01 ^ig pOVA 0.1 pg pOVA 1 jig pOVA
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1000 10000 10 100 1000 10000 1 --- CFSE 10 100 1000 10000 1 < >o
1000 10000Figure 4.8 B
(figure legend on previous page)n (D 5 a cr (U n 0
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en o\ Ul4.2.4 Stringent Requirements of DCs for Cognate T-Cell Feedback for the