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The Journal of Immunology, 2008, 181, 5974 -5980
Copyright © 2008 by The American Association of Immunologists, Inc.
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Heterospecific CD4 Help to Rescue CD8 T Cell Killers1

Marie-Ghislaine de Goër de Herve2,*, Anne Cariou2,*, Federico Simonetta* and Yassine Taoufik3,*,{dagger},{ddagger}

* Institut National de la Santé et de la Recherche Médicale, {dagger} Unite d’Immunologie Biologique, Hôpital de Bicêtre, and {ddagger} Faculté de Médecine, Université Paris 11, Le Kremlin-Bicêtre, France


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Help from CD4 T cells may be required for optimal generation and maintenance of memory CD8 T cells and also for optimal Ag reactivation. We examined whether the helper cell and the CD8 killer cell need to have the same Ag specificity for help to be effective during interactions of memory T cells with mature APC. This is important because virus and tumor Ag-specific CD4 T cell responses are selectively impaired in several chronic viral infections and malignancies. We performed studies in vitro and in vivo and found that functional memory CD4 T cells generated from a distinct antigenic source (heterospecific helpers) could provide direct and effective help to memory CD8 T cells. Functional heterospecific memory CD4 T cells could also rescue secondary CD8 T cell responses in an experimental tumor model in which homospecific CD4 help was impaired. This could provide a rationale for immunotherapy strategies designed to bypass impaired homospecific help.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The CD4 T cells play a pivotal role in coordinating immune responses, providing critical regulation signals by releasing cytokines such as IL-2, and through cognate interactions such as CD40 ligation, with key players of the adaptative immune response, such as dendritic cells (DCs),4 B cells, and cytotoxic CD8 T cells. Although CD4 help is not required for all primary CD8 T cell responses, particularly in inflammatory environments, its loss results in defective CD8 T cell memory responses (1). The mechanisms of CD4 help are largely unknown. Recent results suggest that memory CD8 T cells lacking CD4 help express TRAIL on Ag restimulation and die by TRAIL-mediated cell death (2, 3). Signals from CD4 T cells appear to be necessary during initial Ag activation of naive CD8 T cells (2, 4). The presence of CD4 T cells could also be required for the long-term maintenance of memory CD8 T cells after Ag is cleared (5). Other studies have also indicated deficiencies in secondary CD8 T cell responses in the absence of CD4 T cells at the time of Ag reactivation of memory CD8 T cells (6, 7, 8, 9, 10).

One interesting question is whether the helper cell and the CD8 killer cell need to have the same Ag specificity. The help provided by CD4 T cells for long-term maintenance of CD8 memory T cells is likely Ag-nonspecific (5). However, the situation is less clear in the case of help provided during Ag activation of CD8 T cells by DCs, during priming of naive cells, or re-stimulation of memory cells. This has potential therapeutic implications because specific CD4 T cell responses are selectively impaired in several chronic viral infections and also malignancies, both in humans and in experimental models (7, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22). This impairment may lead to suboptimal memory CD8 T cell responses and thus to Ag persistence. Bypassing defective Ag-specific CD4 help by providing functional help from CD4 T cells of other Ag specificities which are not under the same tolerance phenomena could be one potential immunotherapy approach. In this study, we examined this issue.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

All the mouse strains were on a C57BL/6 background. Wild-type C57BL/6 mice were purchased from Janvier. CD3{epsilon}–/– mice (23) were purchased from CDTA. Marilyn and Matahari mice were kindly provided by Dr. O. Lantz (Institut Curie, Paris). Marilyn mice (24) carry a transgenic CD4 TCR specific for the I-Ab-restricted male Dby peptide NAGFNNRANSSRSS. Matahari mice (25) carry a CD8 TCR transgenic for the male Ag (H-Y)-derived immunodominant peptide WMHHNMDLI (from the Uty gene), complexed with the MHC class I molecule H-2Db. OT-II mice (Charles River Laboratories) carry a transgenic CD4 TCR specific for the I-Ab-restricted OVA peptide aa 323–339 (OVA (323–339)). All TCR-transgenic mice were on a RAG–/– background. All the protocols were approved by our local ethics committee (Villejuif, France).

Immunization protocols

In in vitro experiments shown in Fig. 1, eight virgin female wild-type C57BL/6 mice aged 6–12 wk were divided into two groups and received either 1) i.p. injection of 150 µg of purified OVA emulsified in CFA (Sigma-Aldrich) and, 2 wk later, 150 µg of purified OVA emulsified in IFA; or 2) two i.p. injections, at 2-wk intervals, of 2 x 106 male splenocytes in 100 µl of HBSS. Two weeks later, the mice were killed and splenocytes were pooled in each group. In some experiments, mice were immunized with male splenocytes plus CFA then 2 wk later male splenocytes plus IFA. Similar results as those shown in Fig. 1 were found (not shown).


Figure 1
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  		FIGURE 1. Heterospecific CD4 T cells provide effective help in vitro. CD8 T cells isolated from male-Ag-vaccinated mice (H-Y) were cocultured with CD4 T cells isolated from male-Ag- (H-Y) or OVA-vaccinated (OVA) female mice and mature DCs presenting male-cell and OVA Ags for 5 days. A, Proliferating cells among CD8Uty+ cells. Results are mean ± SEM of three independent experiments. Each experiment was performed in duplicate. B, CD107 exposure on the surface of CD8Uty+ cells was measured by flow cytometry after challenge with male target splenocytes. Results are the mean ± SEM of six independent experiments. C, Granzyme B Elispot assay after challenge with male target splenocytes. Results are the mean ± SEM of three independent experiments.

 
Cell lines, cells, and culture

The Lewis lung carcinoma (LLC) cell line was kindly provided by Dr. C. Denis (INSERM U770, Le Kremlin-Bicêtre, France). LLC cells were cultured in DMEM containing 10% FCS. To prepare LLC-tumor lysates, tumors were dissociated and cells were lysed by three freeze-thaw cycles. Primary cells were cultured in RPMI 1640 medium containing 10% FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, and 50 µM 2-ME (hereafter referred to as complete medium). Male apoptotic cells, obtained after osmotic shock, were used as a source of male Ag. DCs were isolated from spleens of untreated mice (anti-CD11c Microbeads; Miltenyi Biotec). Bone marrow-derived DCs (BMDCs) were differentiated from BM progenitors after culture in GM-CSF-containing medium (R&D Systems), as previously described (26).

CD4 and CD8 T cells were isolated from splenocytes (CD4 and CD8 Particles DM; BD Biosciences). For T cell-DC coculture, CD4 and CD8 T cells were purified from male-immunized spleens, and CD4 T cells were purified from OVA-immunized spleens. Splenic DCs were loaded with male apoptotic cells (2 x 106 apoptotic cells/1 x 106 DCs) and/or OVA (2 µg/ml) and matured with 0.5 µg/ml LPS (Sigma-Aldrich) for 24 h. DCs were then washed extensively before use. One hundred thousand DCs were cocultured with 1 x 106 CD8 T cells in the presence or absence of 1 x 106 CD4 T cells in 1 ml of complete medium for 5 days.

For skin graft experiments, immature BMDCs were loaded with 10 µM OVA (323–339) (Neomps) and with 2 x 106 male apoptotic cells/ml. For tumor experiments, immature BMDCs were loaded 3 h with 10 µM OVA (323–339) and/or 100 µg/ml LLC tumor lysate, then washed and matured for 24 h with LPS (0.5 µg/ml). Tumor-infiltrating lymphocytes (TILs) were prepared from tumors after digestion for 90 min in complete medium containing 1 mg/ml collagenase D (Sigma-Aldrich) at room temperature. EDTA 10 mM was added for the last 10 min. After homogenization, the tumor digestion was filtered through a 70-µm cell strainer and cells were resuspended in 50 ml of HBSS.

Skin grafts

All skin-graft recipients were female MataHari or CD3{epsilon}–/– mice. CD4 T cells (5 x 105) purified from female OT-II or Marilyn mice were injected i.v. into recipient mice. Control mice were injected with 100 µl of HBSS. Two days later, recipient mice were vaccinated s.c. in the scruff of the neck, with 5 x 105 BMDCs double-loaded with OVA (323–339) and male apoptotic splenocytes. Three weeks later, mice were grafted on the back with male tail skin on the right (27) and the mouse’s own tail skin on the left. Recipient mice were then vaccinated with 5 x 105 double-loaded BMDCs. The Band-Aid was removed on day 5 postgrafting, and the graft was inspected daily thereafter. Rejection was defined as complete necrosis and loss of viable skin tissue.

Tumor experiments

C57BL/6 mice aged 6–12 wk were vaccinated with 150 µg of purified OVA emulsified in 100 µl of CFA. Two weeks later, the mice were injected s.c. in the left flank with 105 LLC cells. Ten to 12 days later, when the tumor was palpable, the mice were injected s.c. in the right flank with 5 x 105 Ag-loaded mature BMDCs. Two weeks later, the mice received a second injection of Ag-loaded mature BMDCs. The tumor was measured three times a week with a digital caliper. Tumor volume (mm3) was estimated as follows: width x width x length x {pi}/6.

Flow cytometry and ELISPOT

For CD107/dextramer staining, 105 male target cells were added per well to the coculture (see above), together with 1 µl of FITC-labeled anti-CD107a and anti-CD107b (BD Biosciences) and 10 µM monensin (Sigma-Aldrich) for 6 h. The cells were then stained with anti-CD8-PECy5 (eBioscience) and Uty-specific dextramer-PE (Dako). The cells were analyzed by flow cytometry (EPICS XL; Beckman Coulter). Cell surface CD107 expression was analyzed on CD8+Uty+ cells. To detect TILs, 500 µl of tumor cell suspension was stained with combination of the following Abs: CD8-FITC, PD-1-PE, CD4-PC5, Granzyme B-PE, Foxp3-FITC, CD8-PE, and TRAIL-PE (eBioscience). For Granzyme-B staining, cells were permeabilized by the mean of Cytofix/cytoperm kit (BD Biosciences). Results are expressed as cell numbers per 100 mm3 of tumor. For the Granzyme B ELISPOT, serial dilutions of the coculture were incubated for an additional 24 h on an anti-Granzyme-B-coated plate (R&D Systems). ELISPOT was performed as recommended by the manufacturer. Spots were counted with a Zeiss ELISPOT counter (Zeiss). The results are expressed as the number of Granzyme B-producing cells per 106 CD8 T cells plated at the beginning of the coculture. For the cytotoxicity assay, 104 CFSE-labeled LLC cells were cocultured for 20 h with CD8 T cells isolated from the tumor at a 1:10 ratio. Cell apoptosis was analyzed following Annexin-V-PE and 7-AAD labeling.

Proliferation assays

For analysis of CD8 T cell proliferation in Fig. 1A, CD8 T cells were stained with 0.5 µM CFSE and cocultured for 5 days with CD4 T cells and Ag-loaded mature DCs, as described above. On day 5, the cells were stained with anti-CD8-PC5 and Uty-dextramer. CFSE dilution was analyzed in the CD8Uty+ population by using the FlowJo software (Tree Star). This software allowed us to determine the percentage of cells that divided among original CD8Uty+ population. In proliferation experiments performed in Fig. 3, A–C, total splenocytes, total LN cells, or TILs isolated by CD4 or CD8 positive selection were stained with 0.5 µM CFSE and cocultured for 7 days with BMDCs loaded with OVA (323–339) and/or tumor lysate, as described above. On day 7, the cells were stained with anti-CD4-FITC and anti-CD8-PE, and CFSE dilution was analyzed in the CD4+ and CD8+ populations by means of flow cytometry. The results were expressed as the percentage of CFSElow cells on day 7.


Figure 3
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  		FIGURE 3. Replacing deficient anti-tumoral homospecific CD4 help with functional heterospecific CD4 help inhibits tumor growth. A–C, Mice were vaccinated with OVA emulsified in CFA, and a LLC tumor was induced 2 wk later by s.c. injection in the left flank. Two to three weeks later, tumor draining lymph node cells (TDLN), splenocytes and TILs were isolated and stained with CFSE, then CD4 and CD8 T cell proliferation was tested after 7 days of coculture with mature DCs loaded with tumor lysate (DCTum; Fig. 3, A and C) or OVA (323–339) (DCOVA323–339; Fig. 3, B and C) or both tumor lysate and OVA (323–339) (DCOVA323–339+Tum) (Fig. 3C). CD8 T cells proliferation was evaluated only with cells from the tumor-draining lymph node. Results are mean ± SEM (3–7 mice per condition in each graph). D, mice were vaccinated with OVA emulsified in CFA, and an LLC tumor was induced 2 wk later by s.c. injection in the left flank. When the tumor was palpable (10–12 days), mice were injected with 105 mature DCs loaded with tumor lysate alone (DCTum) or with tumor lysate and OVA323–339 (DCOVA323–339+Tum). Two weeks later, mice received a second injection of Ag-loaded mature BMDCs. Tumor size was recorded three times a week. The data correspond to one experiment representative of a total of four independent experiments, with four to five mice per condition per independent experiment.

 
Statistical analysis

The Wilcoxon test and the Spearman Rank test were used.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Heterospecific CD4 T cells can provide effective help in vitro

We first investigated whether, in vitro, heterospecific CD4 T cells could provide effective help to memory CD8 T cells for secondary expansion and effector functions. CD4 T cells were purified from OVA-vaccinated virgin female wild-type mice (CD4OVA) and male-cell-vaccinated virgin female mice (CD4H-Y). CD8 T cells were purified from male-cell-vaccinated virgin female mice (CD8H-Y). CD8H-Y cells were cocultured with CD4OVA or CD4H-Y for 5 days in the presence of mature DCs loaded with both apoptotic male splenocytes and OVA (mDCH-Y + OVA). Cell proliferation was examined by CFSE dilution on Uty (male immunodominant epitope)-specific CD8H-Y cells. As shown in Fig. 1A, the presence of CD4 T cells during CD8 T cell reactivation by mature DCs increased the percentage of proliferating cells. To exert their positive effects, CD4 T cells had to be activated with their specific Ag presented by mature DCs (Fig. 1A). CD8 T cell proliferation was similar whether the help was Ag-homospecific (CD4H-Y) or -heterospecific (CD4OVA) relative to the CD8 T cell Ag specificity (Fig. 1A). To examine effector functions, CD8H-Y cells were challenged with male target splenocytes, following Ag reactivation with mature DCs and CD4 T cells, as described above. Cell surface CD107 expression, a marker of CD8 T cell degranulation, was analyzed by flow cytometry on Uty-specific CD8H-Y cells (CD8H-Y Uty+). As shown in Fig. 1B, the presence of Ag-activated CD4 T cells during lymphocyte reactivation by mature DCs clearly increased the frequency of CD107-expressing CD8H-Y Uty+ cells. The frequency of CD107-expressing cells was similar whether the help was Ag-homospecific (CD4H-Y) or -heterospecific (CD4OVA) (Fig. 1B). We also analyzed Granzyme-B secretion in an ELISPOT procedure after memory CD4 and CD8 T cell activation by mDCs, as described above, followed by CD8H-Y cell challenge with male target cells. As shown in Fig. 1C, helped CD8H-Y cells secreted higher levels of Granzyme B than unhelped CD8H-Y cells. Help was effective whether or not CD4 T cells were of the same Ag specificity as CD8 T cells, although Granzyme B-secreting CD8H-Y cells were consistently more numerous (however, the difference was not statistically significant, p = 0.247) when provided with homospecific help (Fig. 1C). The low level of specific proliferation and effector functions observed when CD4OVA and CD8H-Y were cocultured with mDCH-Y ruled out the possibility that the positive effects of CD4OVA were related to cross-reactivity with male Ags (Fig. 1, A–C). Together, these results suggest that heterospecific memory CD4 T cells interacting antigenically with the same APC as CD8 T cells can efficiently replace homospecific memory CD4 helpers. They also show that CD4 help is required at the time of Ag reactivation of memory CD8 T cells by mature DCs for optimal secondary expansion and differentiation into effectors.

Heterospecific CD4 T cells can provide effective help in vivo

We also used a skin graft model to globally examine whether heterospecific CD4 T cell help was operational in vivo. TCR transgenic CD4 T cells specific for H-Y (CD4tgH-Y; specific for Dby peptide) or for OVA (323–339) (CD4tgOVA) were adoptively transferred into TCR transgenic female Matahari mice (CD8 T cells of which are specific for the male Ag-derived Uty peptide, CD8tgH-Y). No cross-reactivity of CD4tg-OVA cells toward male Ags was found (Fig. 2A). Two days following adoptive transfer, mice were vaccinated s.c. with mature DCs loaded with both male Ag and OVA I-Ab-restricted peptide (mDCH-Y+ OVA323–339) to prime naive cells. Three weeks after vaccination, mice were revaccinated with mDC H-Y+ OVA323–339 and grafted with male tail skin. As shown in Fig. 2B, grafts survived significantly longer in mice without CD4 help. In mice with CD4 help, the time to graft rejection was similar whether or not the help was Ag-specific (Fig. 2B). At the time of graft, CD4(tgH-Y) and CD4(tgOVA) cells in the corresponding reconstituted Matahari mice represented 28.5 ± 3.66% and 28.2 ± 0.33% of CD3 T cells and 1.03 ± 0.51% and 2.1 ± 0.1% of total splenocytes, respectively (p = 0.921 and p = 0.633, respectively, n = 3 in each group). At the time of graft rejection, CD4(tgH-Y) and CD4(tgOVA) cells represented 22.9% ± 3.6 and 29.8% ± 2.1 of CD3 T cells and 1.12% ± 0.16 and 1.28% ± 0.09 of total splenocytes, respectively (p = 0.158 and p = 0.410, respectively, n = 3 in each group). Controls, consisting of CD3{epsilon}–/– mice adoptively transferred with CD4 (tgOVA) cells, showed no graft rejection, confirming that heterospecific CD4 T cells promoted graft rejection through their helper effect on male-specific CD8 T cells. Together, these results showed that Ag-heterospecific CD4 T cells can directly provide effective help to CD8 T cells in vivo.


Figure 2
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  		FIGURE 2. Heterospecific CD4 T cells provide effective help in vivo too. A, CD4 T cells purified from OT-II transgenic mice were cocultured for 5 days with mature dendritic cells loaded with either male Ag or OVA (323–339) peptide. Proliferation was evaluated by CFSE dilution. B, CD4 T cells from Marylin (CD4 tgH-Y) or OT-II (CD4 tgOVA) mice were adoptively transferred into Matahari (CD8 tgH-Y) or CD3{epsilon}–/– mice, which were then vaccinated with mature DCs loaded with both male and OVA Ags (DCH-Y + OVA). The mice were then grafted with male tail skin, again vaccinated with DCH-Y + OVA, and monitored for graft rejection. Autograft control: mouse’s own tail-skin. Results are mean ± SEM, with five to nine mice per condition.

 
Heterospecific CD4 help improved secondary CD8 T cell responses in a pathological situation in which homospecific help was selectively impaired

Anti-tumoral CD4 T cell responses are impaired in cancer models (15, 16, 18, 19, 21, 22) potentially leading to suboptimal anti-tumoral CD8 T cell responses. We used the well-characterized LLC mouse tumor model, which expresses MHC class I but not class II molecules (not shown). Mice immunized against OVA then injected with LLC cells 2 wk later developed a poorly metastatic s.c. tumor within 2 wk. Four weeks after tumor cell injection, splenic, tumor-draining lymph node, and tumor-infiltrating CD4 T cells showed no significant proliferation in response to mature DC loaded with tumor Ag (mDCTum) (Fig. 3A). By contrast, detectable CD4 T cell responses against OVA were found (Fig. 3B). This suggested selective impairment of tumor-specific CD4 help. CD8 T cells isolated from the tumor-draining lymph node showed no detectable proliferation in response to mDCTum (Fig. 3C). However, mature DCs loaded with both OVA (323–339) and tumor Ag (mDCOVA323–339+Tum) triggered detectable CD8 T cell proliferation (Fig. 3C). We therefore examined whether OVA-specific memory CD4 T cells could replace impaired anti-tumoral CD4 help and thereby improve anti-tumoral CD8 cytotoxic responses and reduce tumor growth in vivo. MDCOVA323–339+Tum or mDCTum cells were injected in the flank opposite to the tumor. This heterospecific help led to a significant reduction in tumor growth compared with untreated mice or mice treated with mDCTum (Fig. 3D). At sacrifice, the tumors were 60% smaller, on average, than in controls (Fig. 3D).

Tumor-infiltrating CD8 T cells and Granzyme B+ CD8 T cells were more numerous in mice treated with mDCOVA323–339+Tum (Fig. 4, A and B). Also, the proportion of Granzyme B+ cells among CD8 T cells was higher than in controls (Fig. 4C). Tumor size at the time of sacrifice correlated negatively with both the total number of CD8 T cells and the number of Granzyme B+ CD8 T cells infiltrating the tumor (Fig. 4, A and B). We then isolated tumor-infiltrating CD8 T cells and challenged them with tumor cells. The ex vivo cytotoxic activity of tumor-infiltrating CD8 T cells was enhanced in mice receiving heterospecific help (Fig. 4D).


Figure 4
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  		FIGURE 4. Heterospecific CD4 help enhances in situ anti-tumoral CD8 T cell response: Mice were treated as described in Fig. 3D. Three weeks following DC injection, tumors were analyzed for CD8 T cell infiltration. A, Absolute number of CD8 T cells/100 mm3 of tumor. B, Absolute number of Granzyme B+ CD8 T cells/100 mm3 of tumor. C, Percentage of Granzyme B+ cells among infiltrating CD8 T cells (mean ± SEM of three independent experiments, six mice per group per experiment). D, Tumor-infiltrating CD8 T cells were isolated and tested for cytotoxicity ex vivo. CD8 T cells were incubated with CFSE-labeled LLC cells for 20 h. Apoptosis was then analyzed in the CFSE+ cell population (LLC) (mean ± SEM of two independent experiments, three mice per group per experiment). E, Percentage of PD-1+ cells among infiltrating CD8 T cells (mean ± SEM of three independent experiments, three mice per group per experiment). F, Percentage of Foxp3+ cells among infiltrating CD4 T cells (mean ± SEM of two independent experiments, three mice per group per experiment). G, TRAIL+ cells among infiltrating CD8 T cells (mean ± SEM of two independent experiments, three mice per group per experiment). H, Percentage of apoptotic cells among infiltrating CD8+ T cells (mean ± SEM of two independent experiments, three mice per group per experiment).

 
Thus, heterospecific CD4 help quantitatively and qualitatively enhanced in situ anti-tumoral CD8 T cell responses and inhibited tumor growth. As PD-1 expression has been implicated in functional inactivation of CD8 T cells in models of chronic viral infection (28), we examined whether heterospecific help enhanced tumor-infiltrating CD8 T cell activity through PD-1 regulation. Although the absolute number of PD-1+ tumor-infiltrating CD8 T cells was increased in mice treated with mDCOVA323–339+Tum, the proportion of PD-1+ cells among tumor-infiltrating CD8 T cells did not differ among the treatment groups (Fig. 4E). FoxP3 is a marker of regulatory T cells. FoxP3 expression was almost undetectable in tumor-infiltrating CD8 T cells (not shown), while the proportion of FoxP3+ cells among tumor-infiltrating CD4 T cells was similar whether or not mice received heterospecific help (Fig. 4F). Also the absolute number of FoxP3+ CD4 T cells infiltrating the tumor did not vary significantly (p = 0.645) (not shown). This indicated that the intratumoral balance between regulatory CD4 T cells and non-regulatory T cells was not affected. Helpless tumor-infiltrating CD8 T cells (untreated mice or mice that received mDCTum) expressed higher levels of the apoptotic ligand TRAIL at their cell surface (Fig. 4G). Apoptosis of helpless tumor-infiltrating CD8 T cells was also higher (Fig. 4H). This supported a previous observation suggesting that regulation of TRAIL expression by CD8 T cells is involved in the mechanisms of CD4 help (3). However, the key point here is that heterospecific help, provided when the tumors were already palpable, was able to restore the helpless secondary anti-tumoral CD8 T cell response in several respects, including TRAIL overexpression. Taken together, our results suggest that heterospecific help can rescue helpless secondary anti-tumoral CD8 T cell responses by improving cell survival, expansion, and cytotoxic functions.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
We show here that heterospecific memory CD4 T cells can provide direct and effective help to memory CD8 T cells interacting with the same mature DC. Heterospecific functional memory CD4 T cells can also rescue secondary CD8 T cell responses in situations in which homospecific CD4 helpers are absent or functionally defective.

Our results do not support previous suggestions that CD4 and CD8 T cells specific for the same Ag presented by the same DC are required for effective help (29, 30). Noteworthy, those conclusions were mainly based on indirect evidence obtained by depleting homospecific helpers without Ag activation of heterospecific CD4 T cells (30). What role might heterospecific help play in physiological conditions? Ag-homospecific helpers are likely to play a prominent role, as suggested by the defective CD8 T cell responses observed in infections and cancer when homospecific CD4 T cell responses are impaired. This could be related to the fact that, in physiological conditions, a given APC is unlikely to co-present Ags derived from distinct pathogens. However, heterospecific help to self-Ags could be involved in self-tolerance breaking and in the onset of autoimmunity. This might be involved in the mechanisms underlying the well-established association between autoimmunity and infections (31).

In an experimental tumor model in which functional anti-tumoral CD4 T cell responses were undetectable, heterospecific CD4 help enhanced anti-tumoral CD8 T cell responses, as reflected by stronger tumor infiltration by CD8 T cells with enhanced cytotoxicity and proliferative capacity. This led to a clear reduction in tumor growth. Mature DC were loaded with a single MHC class II-restricted OVA peptide in addition to a tumor lysate to limit OVA and anti-tumoral CD8 T cell potential competition at the level of the DC. This also clearly showed that the enhanced antitumoral CD8 T cell response was triggered by heterospecific CD4 help.

We cannot rule out the possibility that heterospecific CD4 T cells also provided positive regulatory signals to anti-tumoral CD4 T cells (32, 33), thereby enhancing the antitumoral effect, although the proportion of regulatory T cells among infiltrating CD4 T cells did not vary significantly. Interestingly, tumor-infiltrating CD8 T cells from mice receiving heterospecific help expressed lower levels of the proapoptotic ligand TRAIL and the proportion of apoptotic cells among them was lower. This is consistent with previous observations showing that helpless memory CD8 T cells have a stronger tendency than their helped counterparts to undergo apoptosis on Ag restimulation (3). Among our observations, a critical point is that helpless memory CD8 T cells exhibit a defect in granzyme B-mediated cytotoxic functions. In this respect, a recent observation suggested that TRAIL expression on CD8 T cells negatively regulates the granzymeB/perforin-mediated CD8 T cell killing (34). This suggests that heterospecific CD4 help to memory CD8 T cell may include a TRAIL-dependent component that could control not only survival but also cytotoxic functions. In contrast, we found no significant effect of heterospecific help on tumor-infiltrating CD8 T cell expression of PD-1, a negative regulator of activated T cells that is markedly up-regulated on the surface of exhausted virus-specific CD8 T cells (28). This is consistent with the princeps observation that PD-1 expression on CD8 T cells is independent of CD4 T cells (28) and points to the existence of several pathways that can influence the fate of memory CD8 T cell responses.

These results could provide, at least in part, an immunological explanation for previous observations in humans and experimental cancer models in which the addition of nontumoral Ags (mainly keyhole limpet hemocyanin, an immunogenic carrier protein) to specific immunotherapies enhanced antitumoral activity (35, 36, 37). It is also noteworthy that intravesical immunotherapy with bacillus Calmette-Guérin is an effective adjuvant treatment for bladder cancer. This bacillus Calmette-Guérin effect may involve a mechanism similar to the heterospecific help we describe here. Finally, our results also provide insights into the precise nature of CD4 T cell help required to further improve the clinical efficacy of tumor-specific CD8 T cells (38). Is nonspecific help the best way of promoting CD8 T cell responses, or would protein-specific T cell help, using the CTL epitope and the helper epitope derived from the same protein, be more effective? Clearly, our results suggest that the use of heterospecific help would be more effective in anti-tumoral therapeutic vaccination. Our results and others suggest that anti-tumoral homospecific helpers are selectively impaired in a range of malignancies (15, 16, 18, 19, 21, 22). Moreover, we found that heterospecific memory CD4 T cells are capable to effectively rescue helpless anti-tumoral CD8 T cell responses. It remains to be tested whether heterospecific help could also rescue helpless anti-viral CD8 T cells in chronic viral infections such as hepatitis C virus infection in which a similar selective defect of homospecific help has been reported (7).

Not all Ags have the same fate in vivo. Many of them generate immune responses that lead to their clearance, leaving behind highly functional memory T cells, while others trigger Ag-specific T cell tolerance mechanisms that allow them to persist in the long term. Tolerance processes may target the CD4 T cell, a key regulator of immune responses. In such situations, approaches based on heterospecific CD4 T cell rescue of helpless secondary CD8 T cell responses could be worth trying. Heterospecific help could be provided by stimulating pre-existing functional CD4 T cell memory responses selected in a given patient by in vitro testing of CD4 T cell reactivity against recall Ags (viral, fungal, or mycobacterial Ags).

Mature DCs internalize Ags inefficiently. In vitro loading of monocyte-derived immature DCs with appropriate specific Ags, followed by maturation before administration to patients, would limit the risk of specific autoreactive T cell activation. However, a risk of autoimmunity through activation, in lymphoid tissues, of tolerogenic endogenous immature DCs or autoreactive T cells, via produced cytokines, cannot be ruled out.

In conclusion, we provide evidence that functional heterospecific CD4 T cells can provide effective help to CD8 T cells. This could provide a general framework for effective immunotherapy in situations in which homospecific helpers are impaired.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
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 by grants from Association pour la Recherche contre le Cancer and Académie Nationale de Médecine. Back

2 M.-G.G.H. and A.C. contributed equally to the work. Back

3 Address correspondence and reprint requests to Dr. Y. Taoufik, Institut National de la Santé et de la Recherche Médicale U-802, Faculté de Médecine Paris 11, 63 Rue Gabriel Péri, 94276 Le Kremlin-Bicêtre. E-mail address: yassine.taoufik{at}u-psud.fr Back

4 Abbreviations used in this paper: DC, dendritic cell; LLC, Lewis lung carcinoma; BMDC, bone marrow-derived DC; TIL, tumor-infiltrating lymphocyte; TRAIL, TNF-related apoptosis-inducing ligand. Back

Received for publication March 28, 2008. Accepted for publication August 31, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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