It has been demonstrated that CD4+ T cells require Ag persistence to achieve effective priming, whereas CD8+ T cells are on “autopilot” after only a brief exposure. This finding presents a disturbing conundrum as it does not account for situations in which CD8+ T cells require CD4+ T cell help. We used a physiologic in vivo model to study the requirement of Ag persistence for the cross-priming of minor histocompatibility Ag-specific CD8+ T cells. We report inefficient cross-priming in situations in which male cells are rapidly cleared. Strikingly, the failure to achieve robust CD8+ T cell activation is not due to a problem with cross-presentation. In fact, by providing “extra help” in the form of dendritic cells (DCs) loaded with MHC class II peptide, it was possible to achieve robust activation of CD8+ T cells. Our data suggest that the “licensing” of cross-presenting DCs does not occur during their initial encounter with CD4+ T cells, thus accounting for the requirement for Ag persistence and suggesting that DCs make multiple interactions with CD8+ T cells during the priming phase. These findings imply that long-lived Ag is critical for efficient vaccination protocols in which the CD8+ T cell response is helper-dependent.

Both in vitro and in vivo experimentation have defined an essential role for Ag-specific CD4+ helper cells in the cross-priming of CD8+ T cells (1, 2, 3). This helper activity requires that the Ag be present on the same APC that is engaging the CD8+ T cell, much like the carrier effect that had been previously defined for T cell/B cell cooperation (4). This led to the proposal of three-cell clusters of T cells with APCs, as it could best explain the epitope linkage and noncognate requirements of the in vivo cytolytic response. Due to an awareness that the probability of such interactions would be rare as a result of the low precursor frequency of naive T cells and the scarcity of Ag-loaded APCs, this model gave way to what is commonly referred to as the “serial two-cell” model. Accordingly, CD4+ T cells “license” dendritic cells (DCs)3 that in turn prime CD8+ T cells. This terminology was first introduced by Lanzavecchia (5), following from three studies that support the role of CD4+ T cells offering activation signals to the APC that permit them to prime CD8+ T cells (2, 3, 6).

It has also been demonstrated that CD8+ T cell priming may occur following a relatively short exposure to Ag. In infection situations such as Listeria monocytogenes, effective primary CD8+ T cell responses require less than 24 h of antigenic stimulation (7, 8). Similarly, it has been demonstrated in vitro, using monoclonal T cells that CD8+ T cells given 20 h of stimulus will respond by proliferating extensively and differentiating into effector cells (8, 9). This phenomenon was termed an “autopilot” response by Bevan and Fink (10), and distinguishes CD8+ T cell activation from the regulation of CD4+ T cell priming (11, 12, 13). Indeed, recent experiments from Obst et al. (14) demonstrated that CD4+ T cell proliferation requires cognate stimulation throughout their expansion phase. These results raise a paradox regarding the priming of helper-dependent CD8+ T cell responses: what happens if the DC makes contact with an Ag-specific CD8+ T cell before it is “licensed?” The current model suggests that it would be tolerized, even though the cognate helper cell may be present in the repertoire, albeit late in getting to the Ag-loaded DCs. Moreover, the concept that DCs are licensed by CD4+ T cells during their initial encounter does not account for the need to first prime the CD4+ T cell and induce surface expression of CD40L, a critical molecule for DC activation.

To evaluate the requirement for Ag persistence during helper-dependent cross-priming and address the timing of DC licensing, we studied responses in female mice to the male Ag HY uty peptide, which has the minor histocompatibility locus of Y chromosome in ubiquitously transcribed tetratricopeptide repeat gene uty. In this model, CD8+ T cells that recognize the uty peptide presented by H-2Db are dependent on CD4+ T cell help during the priming phase (6, 15). Moreover, cross-priming to this epitope has been reported to be inefficient in models when allo (H2k→H2b) or β2-microglobulin-deficient (β2m−/−) cells are used as the source of male Ag (16, 17). Finally, the HY model is physiologically and pathologically relevant because the minor histocompatibility Ags are normal cellular proteins and have been implicated in the clinical complications associated with graft-versus-host disease. We report that contrary to the autopilot expansion of CD8+ T cells that has been demonstrated in helper-independent models of CD8+ T cell priming, the priming of HY-reactive CD8+ T cells requires the persistence of Ag.

C57BL/6/J wild-type (WT) mice were obtained from Charles River Breeding Laboratories. PtprcaPepcb/BoyJ (CD45.1), MHC class II−/− (B6.129S-H2dlAb1-Ea/J), and β2m−/− (B6.129P2-B2mtm1Unc/J) mice were obtained from The Jackson Laboratory. MataHari CD8+ TCR transgenic mice (18) were obtained from CDTA (Centre de Distribution Typage et Archivage animal, Orleans, France). All mice were maintained in a Helicobacter-free specific pathogen-free facility, and used under approved protocols.

FACS Abs were obtained from BD Biosciences, Invitrogen, or eBioscience. FACS analysis was performed on a FACSCalibur (BD Biosciences) with the exception of the male cell enrichment studies, which were performed on a FACSCanto II cytometer (BD Biosciences). Abs used in the IFN-γ ELISPOT assays were purchased from Mabtech. Anti-NK1.1 (clone PK136; eBioscience) was used for NK depletion. Recombinant mouse TNF-α was obtained from R&D Systems. The immunodominant H2-Db uty peptide (WMHHNMDLI, accession no. CAA70422, aa 246–254) (19) was provided by NeoMPS. The immunodominant I-Ab dby peptide (NAGFNSNRANSSRSS, accession no. CAA07483, aa 608–622) and OVA peptide (KISQAVHAAHAEINEAG, NP990483, aa 323–339) were provided by Invitrogen Life Technologies. The peptide dby (DEAD (Asp-Glu-Ala-Asp) box polypeptide 3, Y-linked) is used in priming experiments. Labeling with carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) was performed using the Vybrant CFDA-SE cell tracer kit from Molecular Probes.

Splenocytes used for immunization were isolated from male mice. CD11c+ cells were depleted using the Miltenyi MACS system. Of note, the removal of DCs from the injectate ensured that uty protein had to gain access to a host DC as the uty epitope is dependent on expression of the immunoproteasome (20). Immunization was conducted via the intradermal route for the experiments shown, but similar results (as shown in Fig. 1 A) were obtained when using s.c. and i.p. routes for immunization. For NK depletion experiments, we also depleted the injected splenocytes of NK cells to be sure that opsonized cells were not interfering in the priming response (21). NK depletion of mice was achieved by repeated injections of 100 μg of Ab delivered i.p. on the day before and the day of immunization.

FIGURE 1.

Efficient cross-presentation of β2m−/− male cells does not permit effective cross-priming of H-2Db uty-specific CD8+ T cells. A, Female mice were immunized intradermally with 5 × 106 CD11c-depleted splenocytes that had been isolated from WT or β2m−/− male mice. Live, nonclumped, lineage-positive cells were gated. Depletion was confirmed using FACS analysis. B, After 12 days, the efficiency of priming uty-specific CD8+ T cells was determined using IFN-γ ELISPOT. Purified CD8+ T cells were restimulated ex vivo for 24 h using female DCs loaded with uty peptide (•). Unpulsed DCs served as a negative control (○). Each mouse is represented and the number of SFC per 106 total CD8+ T cells is measured. Horizontal bars indicate the mean. This experiment is representative of eight similar experiments. C, To monitor cross-presentation of injected male Ag, the triggering of MataHari CD8+ T cell division was assessed. A total of 2 × 105 CFDA-SE-labeled MataHari CD8+ T cells (CD45.2) was transferred into recipient female mice (CD45.1), and T cell division was followed 3 days after immunization with β2m−/− male cells by gating on CD8β+CD45.2+ cells. Results are representative of four independent experiments from which 3/3 mice tested (1, 2, 3) are in one such experiment.

FIGURE 1.

Efficient cross-presentation of β2m−/− male cells does not permit effective cross-priming of H-2Db uty-specific CD8+ T cells. A, Female mice were immunized intradermally with 5 × 106 CD11c-depleted splenocytes that had been isolated from WT or β2m−/− male mice. Live, nonclumped, lineage-positive cells were gated. Depletion was confirmed using FACS analysis. B, After 12 days, the efficiency of priming uty-specific CD8+ T cells was determined using IFN-γ ELISPOT. Purified CD8+ T cells were restimulated ex vivo for 24 h using female DCs loaded with uty peptide (•). Unpulsed DCs served as a negative control (○). Each mouse is represented and the number of SFC per 106 total CD8+ T cells is measured. Horizontal bars indicate the mean. This experiment is representative of eight similar experiments. C, To monitor cross-presentation of injected male Ag, the triggering of MataHari CD8+ T cell division was assessed. A total of 2 × 105 CFDA-SE-labeled MataHari CD8+ T cells (CD45.2) was transferred into recipient female mice (CD45.1), and T cell division was followed 3 days after immunization with β2m−/− male cells by gating on CD8β+CD45.2+ cells. Results are representative of four independent experiments from which 3/3 mice tested (1, 2, 3) are in one such experiment.

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At 12 days after immunization, spleens were harvested, and CD8+ T cells were purified using anti-CD8 microbeads and MS column (Miltenyi Biotec). ELISPOT assays for IFN-γ-producing cells were performed as previously described (22). The ELISPOT plate evaluation was performed in a blinded fashion by an independent evaluation service (Zellnet Consulting) using an automated ELISPOT reader (Carl Zeiss). For tetramer analysis, soluble MHC/peptide tetramers were produced using a modified version of that described by Altman et al. (23). Baseline staining and the threshold for tetramer reactivity were performed on unimmunized female mice.

Draining lymph nodes (DLN), nondraining lymph nodes, and splenocytes were harvested and genomic DNA was extracted. Mouse Y chromosome-specific DNA was quantified relative to β-actin using the following primers: HY (forward) 5′-AGACAAGTTTTGGGACTGGTGAC-3′ and (reverse) 5′-AGCCCTCCGATGAGGCTGATA-3′; and β-actin (forward) 5′-AGACAAGTTTTGGGACTGGTGAC-3′ and (reverse) 5′-AGCCCTCCGATGAGGCTGATA-3′. Quantitative PCR was performed on genomic DNA using the SYBR Green JumpStart Taq ReadyMix according to the manufacturer’s instructions (Sigma-Aldrich). The reactions were run on a PTC200 equipped with a Chromo4 detector (MJ Research). All the measures were performed in duplicate and validated when the difference in threshold cycle (Ct) between the two measures was less than 0.3. Amplification and dissociation curves displayed a single peak, ruling out the presence of primer dimers (data not shown). The ratio of gene of interest to housekeeping gene was calculated according to the formula ratio = 2−ΔCt, in which ΔCT is mean threshold cycle of gene − mean threshold cycle of housekeeping gene. Ratio was normalized to a standard curve, consisting of 102, 103, 104, 2 × 104, 105, and 5 × 105 male splenocytes in a total of 5 × 106 female splenocytes.

For these experiments, CD45.2 male cells were injected into CD45.1 female hosts. After 2 or 12 days, leukocytes were harvested from 13 lymph nodes (2 inguinal, 2 axillary, 2 brachial, 4 cervical (deep and superficial), 2 para-aortic, and the mesenteric chain) and the spleen, yielding a total of over 1.5 × 108 cells. Anti-CD45.2-PE Ab (eBioscience) followed by labeling with anti-PE Ab linked to iron particles (Miltenyi Biotec) were used to label and enrich the male cells using a MACS column (Miltenyi Biotec). Bound and unbound cells were labeled with anti-CD45.1-FITC (BD Biosciences), a mix of Abs to lineage markers (CD3, CD11b, CD11c, NK1.1, B220, and F4/80) all PacBlue-conjugated (eBioscience). Just before analysis, propidium iodide was added as a marker for dead cells. Approximately 1.5 × 106 cells were recovered from the bound fraction, and all cells were analyzed using a FACSCanto II (BD Biosciences). Live, nonclumped, lineage-positive cells were gated, and the CD45.2+ male cells were enumerated. Total cell numbers were used to determine the absolute number of male cells that persisted in female hosts. Note, this protocol was adapted from the tetramer enrichment studies of Moon and colleagues (24, 25).

MataHari (transgenic animals on a CD45.2 C57BL/6 background) CD8+ T cells were isolated from spleen and lymph nodes and labeled with 5 μM CFDA-SE in PBS. After extensive washing in ice-cold PBS, 5 × 105 MataHari CD8+ T cells were injected i.v. into CD45.1 immunized female recipients. At 3 days after injection, the DLN (regional to the site of immunization) and the spleen were collected. Organs were processed independently, and cells were labeled with CD8β–PE and CD45.2-allophycocyanin Abs allowing for the identification of the transferred MataHari CD8+ T cells and the determination of CFDA-SE intensity.

We hypothesized that the failure to achieve efficient cross-priming of male Ag was due to rapid clearance of the male β2m−/− cells after transfer into female hosts due to activation of NK cells (17). To test this possibility and to determine the requirements for cross-priming CD8+ T cells, we established assays to discriminate the priming of H2-Db-restricted HY uty-specific (referred to in our study as uty) CD8+ T cells and the presentation of the uty peptide. To study CD8+ T cell priming, we injected by intradermal route 5 × 106 male cells into the flank of female hosts. Male splenocytes were depleted of CD11c+ cells to prevent direct presentation of the uty epitope to female CD8+ T cells. Depletion of DCs was confirmed by FACS analysis (Fig. 1,A). It also bears mentioning that the protein under investigation requires processing by the immunoproteosome and thus under sterile, noninflammatory conditions none of the injected cells may themselves generate H-2Db-uty complexes (20). At 12 days after immunization, CD8+ T cells were isolated (>98% purity) and restimulated using uty peptide-loaded DCs. IFN-γ ELISPOT assays were performed as a measure of T cell priming, and data are reported as spot forming cells (SFC) per 106 CD8+ T cells. As previously shown, immunization of female hosts with β2m−/− male splenocytes resulted in inefficient cross-priming as compared with the injection of WT male cells, which induced a robust response (Fig. 1 B).

We next evaluated whether the failure to cross-prime CD8+ T cells following injection of the β2m−/− male splenocytes was due to inefficient cross-presentation. Using CFDA-SE-labeled uty-specific TCR transgenic CD8+ T cells (called Matahari) (18), we demonstrate that in fact cross-presentation of uty Ag results in the vigorous expansion of Ag-specific CD8+ T cells (Fig. 1 C). These data indicate that although the cross-presentation of cell-associated uty is efficient and initial T cell encounter is intact, the indirect pathway does not provide a robust means of priming the endogenous repertoire of female mice.

To evaluate the clearance of male Ag in these priming conditions, we established a quantitative assay to measure the number of male cells that persist in female hosts. Amplification of genomic DNA unique to the Y chromosome allowed for the detection of one male cell within 5 × 105 female cells (data not shown). As expected, WT male cells survived throughout the period of time required for primary expansion and differentiation of the uty-specific CD8+ T cells; however, the β2m−/− male splenocytes were cleared within 6–8 days (Fig. 2 and data not shown). In the experiment shown, we did not observe contraction of the CD8+ T cells after WT male cell immunization, but based on other kinetic studies this phase of the response seems to occur between 21 and 45 days postimmunization (data not shown).

FIGURE 2.

β2m−/− males cells are rapidly cleared after immunization. A, Clearance of β2m−/− male cells was assessed by quantitative measurement of male Y chromosome-specific genomic DNA. WT or β2m−/− male cells were used to immunize female recipients. After 2, 9, 12, and 28 days, leukocytes were isolated from the DLN (shown in this experiment) and spleen (data not shown) and assayed as described in Materials and Methods. A follow-up experiment carefully defined the timing of β2m−/− male cell disappearance as occurring between days 6 and 8 (data not shown). Evaluation of the nondraining lymph node indicated that the β2m−/− male cells were not simply hiding in an alternate lymphoid organ (data not shown). Quantification of the number of male cells was performed and plotted with each individual WT (•, solid line) and β2m−/− (○, dotted line) mouse represented. The limit of detection is a single male cell in 5 × 105 female cells. Lines depicted to connect mean values (horizontal bar). B, Using the remaining cells from mice described in A, uty-tetramer staining was performed to evaluate the expansion of uty-H2-Db specific CD8+ T cells.

FIGURE 2.

β2m−/− males cells are rapidly cleared after immunization. A, Clearance of β2m−/− male cells was assessed by quantitative measurement of male Y chromosome-specific genomic DNA. WT or β2m−/− male cells were used to immunize female recipients. After 2, 9, 12, and 28 days, leukocytes were isolated from the DLN (shown in this experiment) and spleen (data not shown) and assayed as described in Materials and Methods. A follow-up experiment carefully defined the timing of β2m−/− male cell disappearance as occurring between days 6 and 8 (data not shown). Evaluation of the nondraining lymph node indicated that the β2m−/− male cells were not simply hiding in an alternate lymphoid organ (data not shown). Quantification of the number of male cells was performed and plotted with each individual WT (•, solid line) and β2m−/− (○, dotted line) mouse represented. The limit of detection is a single male cell in 5 × 105 female cells. Lines depicted to connect mean values (horizontal bar). B, Using the remaining cells from mice described in A, uty-tetramer staining was performed to evaluate the expansion of uty-H2-Db specific CD8+ T cells.

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To confirm our quantitative PCR-based results using a cell-based methodology, we used congenic markers on the male cells and magnetic enrichment before FACS analysis, which together allowed us to detect 10 male cells in a mixture of 20 × 106 female cells (Fig. 3, A and B). This assay was in concordance with our PCR analysis (Fig. 3,C). For in vivo analysis following immunization with male cells, we harvested the spleen and 13 lymph nodes yielding a total of ∼1.5 × 108 cells. Using this enrichment method, we concentrated the male cells in a total of ∼1.5 × 106 cells and analyzed all cells using a FACSCanto II cytometer. Live, nonclumped cells were identified based on size, scatter, and their negative result for propidium iodide incorporation (Fig. 3,D). From these cells, the lineage-positive cells were positively gated (Fig. 3,D). Female mice injected with 5 × 106 males cells were assayed at days 2 and 12. Results from representative mice are shown (Fig. 3,E) and the absolute number for three mice per group has been plotted for the two time points (Fig. 3 F). A similar absolute number of WT and β2m−/− males cells could be found at day 2; and as described on day 12, we observed the persistence of male cells in the WT group, but not in the mice that had received β2m−/− male cells.

FIGURE 3.

Enumeration of male cells in female mice. An in vitro mixture of CD45.2+ male and CD45.1+ female cells were used to validate our ability to enrich a small number of male cells in the context of female hosts. 0, 20, 200, 2000, or 20,000 male cells were added to 2 × 107 female cells. Samples were split and analyzed separately. A, In the first group, the cell mixture was incubated with anti-CD45.2-PE Ab followed by an anti-PE Ab coupled to an iron particle (Miltenyi Biotec). Enrichment of the CD45.2+ cells was performed using magnetic columns and both the bound and unbound cells were stained using Abs specific for CD45.1 and lineage markers (mixed in a single channel). In addition, propidium iodide was added to exclude dead cells. Live, nonclumped lineage-positive cells are shown, and the gate represents the CD45.2+ cells recovered. B, Data from A are graphically represented, plotting the number of male cells recovered against the input ratio (male cells to female cells) used. The limit of detection was determined to be <1 male cell in 106 female cells. C, Quantitative PCR analysis of the other fraction was performed using the methods described in Materials and Methods. D–F, For in vivo analysis of male cells persisting in female hosts, 13 lymph nodes and the spleen were harvested 2 or 12 days postimmunization with 5 × 106 WT or β2m−/− cells. The gating strategy is shown in D and follows from what is described. E, Representative contour plots are shown for individual mice and gated cells indicate the male cells recovered. F, Data from 3/3 mice per group are plotted with the absolute number of male cells persisting in female mice reported. Individual WT male cells (•) or β2m−/− cells (○) are indicated; bar indicates the average of the population. Data are representative of five experiments.

FIGURE 3.

Enumeration of male cells in female mice. An in vitro mixture of CD45.2+ male and CD45.1+ female cells were used to validate our ability to enrich a small number of male cells in the context of female hosts. 0, 20, 200, 2000, or 20,000 male cells were added to 2 × 107 female cells. Samples were split and analyzed separately. A, In the first group, the cell mixture was incubated with anti-CD45.2-PE Ab followed by an anti-PE Ab coupled to an iron particle (Miltenyi Biotec). Enrichment of the CD45.2+ cells was performed using magnetic columns and both the bound and unbound cells were stained using Abs specific for CD45.1 and lineage markers (mixed in a single channel). In addition, propidium iodide was added to exclude dead cells. Live, nonclumped lineage-positive cells are shown, and the gate represents the CD45.2+ cells recovered. B, Data from A are graphically represented, plotting the number of male cells recovered against the input ratio (male cells to female cells) used. The limit of detection was determined to be <1 male cell in 106 female cells. C, Quantitative PCR analysis of the other fraction was performed using the methods described in Materials and Methods. D–F, For in vivo analysis of male cells persisting in female hosts, 13 lymph nodes and the spleen were harvested 2 or 12 days postimmunization with 5 × 106 WT or β2m−/− cells. The gating strategy is shown in D and follows from what is described. E, Representative contour plots are shown for individual mice and gated cells indicate the male cells recovered. F, Data from 3/3 mice per group are plotted with the absolute number of male cells persisting in female mice reported. Individual WT male cells (•) or β2m−/− cells (○) are indicated; bar indicates the average of the population. Data are representative of five experiments.

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We next wanted a direct test of the observed correlation; in other words it was important to determine whether the lack of Ag persistence accounted for the inefficient priming when β2m−/− male cells were used. As NK cells were the likely cause of death for the β2m−/− splenocytes, we tested the effect of depleting female hosts of NK cells using anti-NK1.1 Ab. NK cell depletion was confirmed by cytometry (data not shown). To avoid potential opsonization by the depleting Ab used for NK depletion, NK cells were removed from the injected male cells. At 12 days postimmunization with β2m−/− male splenocytes, the female host mice were sacrificed and as described, the number of male cells and the frequency of IFN-γ-producing HY-specific T cells were measured. Indeed, NK depletion slowed the clearance of β2m−/− male cells (Fig. 4,A), thus allowing for efficient priming of uty-specific CD8+ T cells (Fig. 4,B). In addition, we confirmed that cross-priming in NK-depleted hosts required CD4+ T cell help (Fig. 4,C). Of note, we have shown that NK depletion of the host does not alter the priming efficiency of CD8+ T cells when using WT male cells for the immunization (data not shown). Together, Figs. 1–4 demonstrate that cross-priming of uty-specific CD8+ T cells is critically dependent on Ag persistence as well as the presence of CD4+ T cell help.

FIGURE 4.

Ag persistence restores efficient cross-priming. To evaluate the effects of delaying Ag clearance, female recipients were depleted of their NK cells by repeated injection of 100 μg of anti-NK1.1 Ab or as a control, injected with similar doses of an isotype matched Ab. WT (A and B) or MHC class II−/− (C) female mice were immunized with β2m−/− male cells. After 12 days, persistent male cells were enumerated (A) and an IFN-γ ELISPOT was performed (B and C). The number of SFC per 106 CD8+ T cells is reported for restimulation with female DCs pulsed with uty peptide. Control wells with unpulsed DCs resulted in fewer than 10 SFC/106 CD8+ T cells and are not shown (B and C). Mean value of three mice is shown, and error bar indicates the SEM. Data are representative of three independent experiments.

FIGURE 4.

Ag persistence restores efficient cross-priming. To evaluate the effects of delaying Ag clearance, female recipients were depleted of their NK cells by repeated injection of 100 μg of anti-NK1.1 Ab or as a control, injected with similar doses of an isotype matched Ab. WT (A and B) or MHC class II−/− (C) female mice were immunized with β2m−/− male cells. After 12 days, persistent male cells were enumerated (A) and an IFN-γ ELISPOT was performed (B and C). The number of SFC per 106 CD8+ T cells is reported for restimulation with female DCs pulsed with uty peptide. Control wells with unpulsed DCs resulted in fewer than 10 SFC/106 CD8+ T cells and are not shown (B and C). Mean value of three mice is shown, and error bar indicates the SEM. Data are representative of three independent experiments.

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Given the data that CD8+ T cells may be primed after only a short contact with cognate MHC class I-peptide complexes (10), it was important to determine whether the need for persistent Ag was in fact functionally linked to the requirement for CD4+ T cell help. In other words, we evaluated whether uty-specific CD8+ T cell cross-priming fails due to the inability to generate a strong HY-specific CD4+ T cell response. WT mice (harboring only the endogenous repertoire) were immunized with β2m−/− male splenocytes while providing sustained stimulation for the HY-specific CD4+ T cells. To achieve this priming condition, we coadministered WT DCs loaded with the immunodominant MHC class II epitope derived from the male Ag HY-dby (DC/dby). As a control, DCs were loaded with an irrelevant MHC class II peptide derived from OVA (DC/ova). The coadministered DCs were i.v. injected to decrease the possibility that they capture the intradermally injected male cells. Notably, the additional stimulation for CD4+ T cells permitted efficient cross-priming of uty-specific CD8+ T cells (Fig. 5,A). Interestingly, the DC/dby but not the DC/ova were capable of providing this “extra help,” suggesting that this effect was dependent on the activation of a population of helper cells that has the ability to engage APC-presenting Ag derived from the β2m−/− male splenocytes. As additional controls for this experiment, we confirmed that the immunization with DC/dby did not alter the kinetics of clearance of the β2m−/− male cells (Fig. 5B and data not shown), and we demonstrated that the effect was dependent on CD4+ T cells as priming did not occur in MHC class II-deficient mice (data not shown).

FIGURE 5.

Efficient cross-priming may be achieved by providing extra help. A, Female mice were immunized with both β2m−/− male splenocytes (delivered intradermally) and 5 × 105 DCs pulsed with the I-Ab-restricted epitope derived from the HY protein dby (i.v. administered). As a test for the requirement of cognate Ag, we also used DCs pulsed with the I-Ab-restricted epitope derived from OVA. Tetramer staining was performed to monitor the expansion of uty-specific CD8+ T cells. Box and Whisker plots are shown and a Mann-Whitney U test was perfomed to calculate the p value. B, Quantification of the number of male cells was performed and average values from three mice are shown with the immunization with male WT splenocytes serving as a positive control.

FIGURE 5.

Efficient cross-priming may be achieved by providing extra help. A, Female mice were immunized with both β2m−/− male splenocytes (delivered intradermally) and 5 × 105 DCs pulsed with the I-Ab-restricted epitope derived from the HY protein dby (i.v. administered). As a test for the requirement of cognate Ag, we also used DCs pulsed with the I-Ab-restricted epitope derived from OVA. Tetramer staining was performed to monitor the expansion of uty-specific CD8+ T cells. Box and Whisker plots are shown and a Mann-Whitney U test was perfomed to calculate the p value. B, Quantification of the number of male cells was performed and average values from three mice are shown with the immunization with male WT splenocytes serving as a positive control.

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To explain how the CD4+ T cells that are primed by the DC/dby influence the cross-priming of CD8+ T cells, we tested whether the injection of DC/dby acts in trans, by increasing the survival of cross-presenting host APCs. To test this possibility, we established an in vivo assay that evaluates the persistence of uty-H2-Db complexes on host APCs, measured as a function of MataHari cell division. As described, female mice were immunized intradermally with either WT or β2m−/− male splenocytes at day 0 of the experiment. CFDA-SE-labeled Matahari were adoptively transferred on the day before immunization (day −1), day 7, or day 11 postimmunization (Fig. 6,A). MataHari proliferation in the DLN was measured on days 3, 10, or 14, respectively, and the percentage of divided MataHari (as a fraction of the total MataHari detected in recipient mice) is reported. As expected, 3 days postimmunization, MataHari proliferation was observed in the DLN for both conditions (Fig. 6,B). By the time of the second MataHari cohort (days 7–10), we observed differences between the WT and β2m−/− conditions, and this difference became much more evident at the third time point (days 11–14). Together with the quantitative analysis of surviving male cells (Fig. 2 A), we conclude that the β2m−/− splenocytes are cleared from female mice by day 8, and the presentation of uty-H2-Db complexes decays soon after, with insufficient levels of expression by day 11 to initiate robust proliferation of MataHari CD8+ T cells.

FIGURE 6.

Persistence of cross-presented class I Ag is extended by extra help. A and B, At day 0, female C57BL/6 mice were immunized intradermally with WT or β2m−/− male CD11c-depleted splenocytes (5 × 106 cells/mouse). Schematic representation of the experiment shows three independent cohorts of CFDA-SE-labeled MataHari T cells were injected i.v. to determine the availability of H-2Db-uty complexes. On days 1, 7, and 11 and 3 days later, the dilution of CFDA-SE in the dividing MataHari T cells was evaluated by FACS. Results are expressed as a percentage of divided MataHari T cells over the total MataHari T cells in the DLN. Each individual mouse is represented, and the horizontal bar represents the mean. C, Using the approach detailed in Fig. 5, we monitored the division of MaraHari T cells in female mice coimmunized with β2m−/− male splenocytes (delivered intradermally) and 5 × 105 DCs pulsed with dby (i.v. administered). Data from the interval 9–12 days after immunization are shown. The percentage of divided MataHari is reported as a percentage of the total MataHari CD8+ T cells. Data from one of two representative experiments are shown. FACS plots are a composite representation of n = 5 mice, normalized to the absolute number of CD8β+ T cells in the DLN.

FIGURE 6.

Persistence of cross-presented class I Ag is extended by extra help. A and B, At day 0, female C57BL/6 mice were immunized intradermally with WT or β2m−/− male CD11c-depleted splenocytes (5 × 106 cells/mouse). Schematic representation of the experiment shows three independent cohorts of CFDA-SE-labeled MataHari T cells were injected i.v. to determine the availability of H-2Db-uty complexes. On days 1, 7, and 11 and 3 days later, the dilution of CFDA-SE in the dividing MataHari T cells was evaluated by FACS. Results are expressed as a percentage of divided MataHari T cells over the total MataHari T cells in the DLN. Each individual mouse is represented, and the horizontal bar represents the mean. C, Using the approach detailed in Fig. 5, we monitored the division of MaraHari T cells in female mice coimmunized with β2m−/− male splenocytes (delivered intradermally) and 5 × 105 DCs pulsed with dby (i.v. administered). Data from the interval 9–12 days after immunization are shown. The percentage of divided MataHari is reported as a percentage of the total MataHari CD8+ T cells. Data from one of two representative experiments are shown. FACS plots are a composite representation of n = 5 mice, normalized to the absolute number of CD8β+ T cells in the DLN.

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Using this assay system, we compared the availability of uty-H2-Db complexes in mice that received β2m−/− male splenocytes, injected alone or in combination with DC/dby. At day 9, a time point in which the cross-presented uty-H2-Db complexes were beginning to wane, we find that the coadministration of DC/dby could preserve Ag presentation as indicated by the increased percentage of divided MataHari cells (Fig. 6 C). In an independent experiment (n = 5 mice per group), we compared the availability of uty-H2-Db complexes in mice that received β2m−/− coadministered with DC/dby vs mice that were immunized with WT male cells. Importantly, the extra help provided by DC/dby offered the necessary signals to achieve a level of uty-H2-Db persistence that was equivalent to what is seen with the injection of WT male cells (data not shown). Based on these results and on the ability of extra help to restore effective CD8+ T cell priming, we conclude that CD4+ T cells act in trans to delay the disappearance of MHC class I-peptide complexes generated by Ag cross-presentation.

These experiments evaluate the question of whether Ag-specific cross-priming depends on the continued engagement of the TCR, or on the conditions in which the Ag is initially encountered. As a final control, we returned to the observation that cross-presentation of uty-derived β2m−/− male cells is robust (Fig. 1,B). In considering this result, we had to clarify why TCR engagement of Ag-specific CD8+ T cells did not result in an autopilot response (10). One important caveat could account for our results: due to an insufficient number of H-2Db-uty complexes, it may not be possible to trigger an autopilot response in the HY system. Again, using adoptive transfer of naive MataHari CD8+ T cells, it was possible to exclude this possibility. It has been shown that by adoptive transfer of a high number of monoclonal T cells, CD8+ T cells are able to help themselves (26, 27). Indeed, this result is true for the HY model as well (Fig. 7,A). As β2m−/− male splenocytes were injected into female mice that had received 105 MataHari CD8+ T cells, it was in fact possible to achieve efficient cross-priming. CD4+ T cell depletion confirmed that with an increased precursor frequency of CD8+ T cells, the priming is helper-independent (Fig. 7 B). Therefore, we conclude that for the endogenous repertoire, in which Ag reactive T cells are rare, cross-priming requires persistent Ag. The persistence is actually playing a dual role: first for the priming of CD4+ Th cells, which in turn prolonged presentation of the MHC class I epitope. Together, these results suggest that DC licensing is a delayed event, thus providing a refined model for how helper-dependent CD8+ T cell priming occurs.

FIGURE 7.

High precursor frequency of HY CD8+ T cells obviates the need for persistent Ag and renders cross-priming helper-independent. A, A total of 105 MataHari T cells were adoptively transferred into female mice. Female C57BL/6 mice were immunized intradermally with WT or β2m−/− male CD11c-depleted splenocytes (5 × 106) as described in Fig. 6. IFN-γ ELISPOT was performed to assess the efficiency of cross-priming. B, Depletion of CD4+ T cells was performed 1 day before the immunization, and CD8+ T cell priming was assessed. Restimulation in the ELISPOT was performed with uty-pulsed DCs (•) and DCs alone (○).

FIGURE 7.

High precursor frequency of HY CD8+ T cells obviates the need for persistent Ag and renders cross-priming helper-independent. A, A total of 105 MataHari T cells were adoptively transferred into female mice. Female C57BL/6 mice were immunized intradermally with WT or β2m−/− male CD11c-depleted splenocytes (5 × 106) as described in Fig. 6. IFN-γ ELISPOT was performed to assess the efficiency of cross-priming. B, Depletion of CD4+ T cells was performed 1 day before the immunization, and CD8+ T cell priming was assessed. Restimulation in the ELISPOT was performed with uty-pulsed DCs (•) and DCs alone (○).

Close modal

The experiments described in this study evaluate the question of whether helper-dependent cross-priming requires the persistence of Ag. We used the weakly immunogenic HY model Ag in which priming of uty-specific CD8+ T cells is dependent on CD4+ T cell help during the priming phase (28). Quantitative PCR was used as a highly sensitive method for the detection of male genomic DNA (Fig. 2,A and data not shown). In addition, we used magnetic enrichment of transferred cells as a means of measuring Ag persistence in its cellular form (Fig. 3). These systems allowed us to dissect the role of Ag as a limiting resource; and we demonstrate that indeed the lack of Ag availability accounts for the inefficient cross-priming of minor histocompatibility Ag-specific T cells (Figs. 1–4). In studying this problem further, we demonstrated that Ag is required for generating an effective helper cell compartment: coadministration of DCs presenting only MHC class II-peptide complexes provided extra help and permitted effective cross-priming of Ag derived from the short-lived β2m−/− splenocytes (Fig. 5). At first look, however, this result did not fit with the data that CD4+ T cells interact with the same APC as the CD8+ T cell, thus breaking with the idea of epitope linkage and upsetting the notion of cognate help. The real surprise, and the solution to this paradox, came when we began tracking the MHC class I-peptide complex on the cross-presenting APC. The presence of an additional stimulus for the CD4+ Th cells resulted in an increased survival of the cross-presenting APC (Fig. 6). These data are supported by the recent observation that CD40 engagement on DCs results in increased survival (29). In this study, exogenous stimulation of CD40 was equated to sustained expression of endogenous Ag. Our work advances this observation and we have integrated their findings into observations that concern helper-dependent CD8+ T cell cross-priming. An alternate, though not favored hypothesis, is that the availability of CD4+ T cells permits serial cross-presentation as this could also account for an increase in half-life of the MHC class I-uty complexes. Thus, we conclude that CD8+ T cells require Ag persistence, not simply for the maintenance of CD4+ Th cells that are serving to license DCs, but perhaps also to complete their own differentiation process, executed by multiple contacts with DCs (Fig. 8).

FIGURE 8.

Cross-priming CD8+ T cells requires extra help. During physiologic situations of priming, DCs present captured Ag to CD8+ and CD4+ T cells. This mechanism results in several rounds of divisions, and for CD8+ T cells, full expansion in the absence of additional TCR engagement is possible. However, in the absence of a licensing signal, this results in an abortive response as the CD8+ T cells undergo apoptosis (a). We demonstrate that for helper-dependent responses, Ag persistence is required, not necessarily for the CD8+ T cells, but also for the priming and maintenance of CD4+ Th cells that serve to license DCs to cross-prime. We propose that this licensing decision is made after an initial round of CD8+ T cell divisions (b), and as such, there may also be a requirement for CD8+ T cells to re-encounter DCs that are harboring cognate Ag. This work challenges the current interpretation of the autopilot response and resolves several conflicting aspects of DC and CD8+ T cell engagement by discriminating initial engagement of the TCR from the licensing step, which is critically dependent on Ag persistence in physiologic responses.

FIGURE 8.

Cross-priming CD8+ T cells requires extra help. During physiologic situations of priming, DCs present captured Ag to CD8+ and CD4+ T cells. This mechanism results in several rounds of divisions, and for CD8+ T cells, full expansion in the absence of additional TCR engagement is possible. However, in the absence of a licensing signal, this results in an abortive response as the CD8+ T cells undergo apoptosis (a). We demonstrate that for helper-dependent responses, Ag persistence is required, not necessarily for the CD8+ T cells, but also for the priming and maintenance of CD4+ Th cells that serve to license DCs to cross-prime. We propose that this licensing decision is made after an initial round of CD8+ T cell divisions (b), and as such, there may also be a requirement for CD8+ T cells to re-encounter DCs that are harboring cognate Ag. This work challenges the current interpretation of the autopilot response and resolves several conflicting aspects of DC and CD8+ T cell engagement by discriminating initial engagement of the TCR from the licensing step, which is critically dependent on Ag persistence in physiologic responses.

Close modal

These data have pushed us to re-evaluate the requirements for naive CD8+ T cells to set in motion a developmental program that includes multiple rounds of division and the acquisition of effector function. Three related studies form the bedrock of the CD8+ T cell autopilot model. In two of the studies, injection of high CFU counts of L. monocytogenes resulted in recruitment of Ag-specific naive CD8+ T cells during the first 24 h (7, 11). Antibiotics were used to kill off the Ag and still, in the absence of further TCR engagement, the responding T cells were capable of dividing and differentiating into effector cytolytic T cells (7). In the third study, using an engineered APC, thus permitting control over the kinetics of antigenic stimulation, the time required to achieve CD8+ T cell programming was carefully determined. As little as 20 h of exposure to APCs can trigger subsequent divisions and differentiation (9). From these studies, it was concluded that after initial encounter with Ag, the priming of CD8+ T cells does not rely on additional TCR engagement nor on CD4+ T cell help (10). These data, however, must be considered in the face of more recent work, as our understanding of helper-dependent CD8+ activation has been radically modified. CD8+ T cells that are primed in the absence of help, fail to respond during secondary restimulation (30, 31). The mechanism for this helper effect may act via the APC, resulting in the programming of naive T cells as supported by Janssen et al. (30). Alternatively, other models support a role for Ag nonspecific CD4+ helper cells that provide survival signals for the maintenance of a high precursor frequency of memory CD8+ T cells (32). What is clear is the need to differentiate the signals involved in CD8+ T cell proliferation from those that govern the acquisition of effector functions. Additionally, these results point out that much of our understanding about the priming of naive T cells was contingent on either the use of TCR transgenic CD8+ T cells or assays that evaluated secondary restimulation.

Two prior studies have concluded that Ag persistence is required for efficient CD8+ T cell priming. Curtsinger et al. (33) evaluated the requirement for priming OVA-specific TCR transgenic T cells. In their in vivo experiments, they adoptively transfer >106 OT-I and prime with peptide as a source of OVA257–264. Although their work offers important information about competition for Ag when the precursor frequency of CD8+ T cells is high, it does not bear on the requirements for priming endogenous T cell responses. In the second study, Storni et al. (34) compared p33 lymphocytic choriomeningitis virus peptide immunization with viral-like particles expressing the p33 peptide fused to the C terminus of hepatitis B c Ag. The results demonstrate that peptide plus CpG fails to maintain robust p14 TCR transgenic T cell proliferation, whereas viral-like particles plus CpG increases CD8+ T cell priming and enhances protection from subsequent vaccinia virus challenge. Based on the short half-life of peptide for the stimulation of CD8+ T cells, the researchers conclude that Ag persistence is an important component for efficient priming. Again, this second model is CD4+ Th cell-independent during the primary CD8+ T cell stimulation (35, 36), and does not address cross-priming. Finally, there is the new study from Tyznik and Bevan (37) concerning the priming of male-specific responses. The report indicates that male CD8+ T cells within the inoculate suppress the priming of female anti-HY CD8+ T cell responses. This result is observed, however, only at high numbers of injected cells (i.v. route); in comparison, we use 4-fold fewer cells (injected intradermally) for immunization and thus do not see evidence for veto suppression in our model system.

So how does our newfound recognition of the importance of CD4+ T cells alter the interpretation of the autopilot model for CD8+ T cell activation? Drawing from the conclusions of our study on helper-dependent cross-priming, it would follow that in the absence of persistent Ag but the presence of a high precursor frequency of responding CD8+ T cells (as was the case for the autopilot studies (7, 9, 11)), CD8+ T cells would divide and differentiate into effector cells; however, secondary engagement would result in an abortive response due to the absence of CD4+ T cells during the priming phase. We conclude that for helper-dependent priming, such as the cross-priming of minor histocompatibility Ag-specific T cells, persistent Ag is required to support the differentiation of effector CD8+ T cells. For the recent study by Prlic et al. (38), we see no conflict because their model system does not have a strict requirement for CD4+ T cell help.

In summary, we propose that under physiologic conditions, CD4+ T cells provide help in a cognate manner and as such, support the creation of a local microenvironment. In particular, the helper cells facilitate the persistence of MHC class I-peptide complexes that provide “signal 1” for cross-priming of CD8+ T cells. Such a model would predict that during physiologic situations of priming, the decision between activation and tolerance may be made after the initial round of CD8+ T cell divisions (Fig. 8). This revised model clarifies the distinct outcomes of the initial contact between DCs and CD8+ T cells. When the outcome is cross-tolerant, we know that CD8+ T cells engage DC cross-presenting tissue-derived Ag, and they undergo several rounds of divisions and proceed to die (1, 26) (Fig. 8). If during this window time period, DCs are licensed by CD4+ T cells, the response may be converted with activation signals being provided to the CD8+ T cells, rescuing them from programmed cell death. By delaying the immunologic decision until a secondary encounter, we offer a revised interpretation for the autopilot response, and with an expanded population of responder T cells, our data may also account for how a rare three-cell interaction is possible under physiologic conditions (39, 40). These results will impact our understanding of graft-versus-host disease and influence vaccination strategies that are aimed at priming CD8+ T cell responses.

We thank Drs. James Di Santo and Lisa Walter for helpful comments and critical review of the manuscript. We also thank the staff of the Institut Pasteur Animal Facility.

The authors have no financial conflict of interest.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

This work was supported in part by grants from Mildred Scheel Stipendium Deutsche Krebshilfe e.V. (to M.U.), La Ligue Nationale Contre le Cancer, L’Agencé Nationale de la Récherche, and The European Young Investigator Awards Scheme, European Science Foundation (to M.L.A.).

3

Abbreviations used in this paper: DC, dendritic cell; CFDA-SE, carboxyfluorescein diacetate succinimidyl ester; β2m, β2-microglobulin; DLN, draining lymph node; SFC, spot forming cell; WT, wild type.

1
Albert, M. L., M. Jegathesan, R. B. Darnell.
2001
. Dendritic cell maturation is required for the cross-tolerization of CD8+ T cells.
Nat. Immunol.
2
:
1010
-1017.
2
Schoenberger, S. P., R. E. Toes, E. I. van der Voort, R. Offringa, C. J. Melief.
1998
. T-cell help for cytotoxic T lymphocytes is mediated by CD40-CD40L interactions.
Nature
393
:
480
-483.
3
Bennett, S. R., F. R. Carbone, F. Karamalis, R. A. Flavell, J. F. Miller, W. R. Heath.
1998
. Help for cytotoxic-T-cell responses is mediated by CD40 signalling.
Nature
393
:
478
-480.
4
Mitchison, N. A., C. O'Malley.
1987
. Three-cell-type clusters of T cells with antigen-presenting cells best explain the epitope linkage and noncognate requirements of the in vivo cytolytic response.
Eur. J. Immunol.
17
:
1579
-1583.
5
Lanzavecchia, A..
1998
. Immunology: licence to kill.
Nature
393
:
413
-414.
6
Ridge, J. P., F. Di Rosa, P. Matzinger.
1998
. A conditioned dendritic cell can be a temporal bridge between a CD4+ T-helper and a T-killer cell.
Nature
393
:
474
-478.
7
Mercado, R., S. Vijh, S. E. Allen, K. Kerksiek, I. M. Pilip, E. G. Pamer.
2000
. Early programming of T cell populations responding to bacterial infection.
J. Immunol.
165
:
6833
-6839.
8
Wong, P., E. G. Pamer.
2001
. Cutting edge: antigen-independent CD8 T cell proliferation.
J. Immunol.
166
:
5864
-5868.
9
van Stipdonk, M. J., G. Hardenberg, M. S. Bijker, E. E. Lemmens, N. M. Droin, D. R. Green, S. P. Schoenberger.
2003
. Dynamic programming of CD8+ T lymphocyte responses.
Nat. Immunol.
4
:
361
-365.
10
Bevan, M. J., P. J. Fink.
2001
. The CD8 response on autopilot.
Nat. Immunol.
2
:
381
-382.
11
Kaech, S. M., R. Ahmed.
2001
. Memory CD8+ T cell differentiation: initial antigen encounter triggers a developmental program in naive cells.
Nat. Immunol.
2
:
415
-422.
12
Bajenoff, M., O. Wurtz, S. Guerder.
2002
. Repeated antigen exposure is necessary for the differentiation, but not the initial proliferation, of naive CD4+ T cells.
J. Immunol.
168
:
1723
-1729.
13
Celli, S., Z. Garcia, P. Bousso.
2005
. CD4 T cells integrate signals delivered during successive DC encounters in vivo.
J. Exp. Med.
202
:
1271
-1278.
14
Obst, R., H. M. van Santen, D. Mathis, C. Benoist.
2005
. Antigen persistence is required throughout the expansion phase of a CD4+ T cell response.
J. Exp. Med.
201
:
1555
-1565.
15
Guerder, S., P. Matzinger.
1992
. A fail-safe mechanism for maintaining self-tolerance.
J. Exp. Med.
176
:
553
-564.
16
Desquenne-Clark, L., H. Kimura, W. K. Silvers.
1992
. Priming and cross-priming for H-Y in female mice.
Transplantation
54
:
916
-919.
17
Millrain, M., P. Chandler, F. Dazzi, D. Scott, E. Simpson, P. J. Dyson.
2001
. Examination of HY response: T cell expansion, immunodominance, and cross-priming revealed by HY tetramer analysis.
J. Immunol.
167
:
3756
-3764.
18
Valujskikh, A., O. Lantz, S. Celli, P. Matzinger, P. S. Heeger.
2002
. Cross-primed CD8+ T cells mediate graft rejection via a distinct effector pathway.
Nat. Immunol.
3
:
844
-851.
19
Greenfield, A., D. Scott, D. Pennisi, I. Ehrmann, P. Ellis, L. Cooper, E. Simpson, P. Koopman.
1996
. An H-YDb epitope is encoded by a novel mouse Y chromosome gene.
Nat. Genet.
14
:
474
-478.
20
Palmowski, M. J., U. Gileadi, M. Salio, A. Gallimore, M. Millrain, E. James, C. Addey, D. Scott, J. Dyson, E. Simpson, V. Cerundolo.
2006
. Role of immunoproteasomes in cross-presentation.
J. Immunol.
177
:
983
-990.
21
Dhodapkar, K. M., J. Krasovsky, B. Williamson, M. V. Dhodapkar.
2002
. Antitumor monoclonal antibodies enhance cross-presentation of cellular antigens and the generation of myeloma-specific killer T cells by dendritic cells.
J. Exp. Med.
195
:
125
-133.
22
Blachère, N. E., R. B. Darnell, M. L. Albert.
2005
. Apoptotic cells deliver processed antigen to dendritic cells for cross-presentation.
PLoS Biol.
3
:
e185
23
Altman, J. D., P. A. Moss, P. J. Goulder, D. H. Barouch, M. G. McHeyzer-Williams, J. I. Bell, A. J. McMichael, M. M. Davis.
1996
. Phenotypic analysis of antigen-specific T lymphocytes.
Science
274
:
94
-96.
24
Hataye, J., J. J. Moon, A. Khoruts, C. Reilly, M. K. Jenkins.
2006
. Naive and memory CD4+ T cell survival controlled by clonal abundance.
Science
312
:
114
-116.
25
Moon, J. J., H. H. Chu, M. Pepper, S. J. McSorley, S. C. Jameson, R. M. Kedl, M. K. Jenkins.
2007
. Naive CD4+ T cell frequency varies for different epitopes and predicts repertoire diversity and response magnitude.
Immunity
27
:
203
-213.
26
Mintern, J. D., G. M. Davey, G. T. Belz, F. R. Carbone, W. R. Heath.
2002
. Cutting edge: precursor frequency affects the helper dependence of cytotoxic T cells.
J. Immunol.
168
:
977
-980.
27
Marzo, A. L., K. D. Klonowski, A. Le Bon, P. Borrow, D. F. Tough, L. Lefrancois.
2005
. Initial T cell frequency dictates memory CD8+ T cell lineage commitment.
Nat. Immunol.
6
:
793
-799.
28
Wang, J. C., A. M. Livingstone.
2003
. Cutting edge: CD4+ T cell help can be essential for primary CD8+ T cell responses in vivo.
J. Immunol.
171
:
6339
-6343.
29
Obst, R., H. M. van Santen, R. Melamed, A. O. Kamphorst, C. Benoist, D. Mathis.
2007
. Sustained antigen presentation can promote an immunogenic T cell response, like dendritic cell activation.
Proc. Natl. Acad. Sci. USA
104
:
15460
-15465.
30
Janssen, E. M., N. M. Droin, E. E. Lemmens, M. J. Pinkoski, S. J. Bensinger, B. D. Ehst, T. S. Griffith, D. R. Green, S. P. Schoenberger.
2005
. CD4+ T-cell help controls CD8+ T-cell memory via TRAIL-mediated activation-induced cell death.
Nature
434
:
88
-93.
31
Sun, J. C., M. J. Bevan.
2003
. Defective CD8 T cell memory following acute infection without CD4 T cell help.
Science
300
:
339
-342.
32
Sun, J. C., M. A. Williams, M. J. Bevan.
2004
. CD4+ T cells are required for the maintenance, not programming, of memory CD8+ T cells after acute infection.
Nat. Immunol.
5
:
927
-933.
33
Curtsinger, J. M., C. M. Johnson, M. F. Mescher.
2003
. CD8 T cell clonal expansion and development of effector function require prolonged exposure to antigen, costimulation, and signal 3 cytokine.
J. Immunol.
171
:
5165
-5171.
34
Storni, T., F. Lechner, I. Erdmann, T. Bachi, A. Jegerlehner, T. Dumrese, T. M. Kundig, C. Ruedl, M. F. Bachmann.
2002
. Critical role for activation of antigen-presenting cells in priming of cytotoxic T cell responses after vaccination with virus-like particles.
J. Immunol.
168
:
2880
-2886.
35
Shedlock, D. J., H. Shen.
2003
. Requirement for CD4 T cell help in generating functional CD8 T cell memory.
Science
300
:
337
-339.
36
Janssen, E. M., E. E. Lemmens, T. Wolfe, U. Christen, M. G. von Herrath, S. P. Schoenberger.
2003
. CD4+ T cells are required for secondary expansion and memory in CD8+ T lymphocytes.
Nature
421
:
852
-856.
37
Tyznik, A. J., M. J. Bevan.
2007
. The surprising kinetics of the T cell response to live antigenic cells.
J. Immunol.
179
:
4988
-4995.
38
Prlic, M., G. Hernandez-Hoyos, M. J. Bevan.
2006
. Duration of the initial TCR stimulus controls the magnitude but not functionality of the CD8+ T cell response.
J. Exp. Med.
203
:
2135
-2143.
39
Castellino, F., A. Y. Huang, G. Altan-Bonnet, S. Stoll, C. Scheinecker, R. N. Germain.
2006
. Chemokines enhance immunity by guiding naive CD8+ T cells to sites of CD4+ T cell-dendritic cell interaction.
Nature
440
:
890
-895.
40
Beuneu, H., Z. Garcia, P. Bousso.
2006
. Cutting edge: cognate CD4 help promotes recruitment of antigen-specific CD8 T cells around dendritic cells.
J. Immunol.
177
:
1406
-1410.