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Normal Responsiveness of CTLA-4-Deficient Anti-Viral Cytotoxic T Cells¹

Martin F. Bachmann^2,*, Paul Waterhouse^*,, Daniel E. Speiser^*, Kim McKall-Faienza^*, Tak W. Mak^*, and Pamela S. Ohashi^*

^* Departments of Medical Biophysics and Immunology, Ontario Cancer Institute; and Amgen Institute, Toronto, Ontario, Canada

	Abstract

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CTLA-4 has been proposed to negatively regulate immune responses, and mice deficient for CTLA-4 expression succumb to a lymphoproliferative disorder within a few weeks after birth. This study assessed the responsiveness of CTLA-4-deficient T cells expressing a class I-restricted TCR specific for lymphocytic choriomeningitis virus (LCMV). The kinetics of T cell proliferation were studied in vitro after stimulation of T cells with full and partial T cell agonists. No gross abnormalities in CTLA-4-deficient T cells could be detected. Using adoptive transfer experiments, T cell responses were also measured in vivo after infection with LCMV. Low dose infection with LCMV leads to strong expansion of specific T cells followed by a reduction in T cells that parallels the elimination of Ag. The kinetics of T cell expansion and elimination after low dose LCMV infection were not affected by the absence of CTLA-4. High dose infection of mice with LCMV leads to a transient expansion of T cells followed by T cell exhaustion, where all specific T cells are eliminated. T cell exhaustion also occurred in the absence of CTLA-4. Thus, surprisingly, the absence of CTLA-4 did not interfere with T cell activation, down-regulation of ongoing T cell responses after the elimination of Ag, or the exhaustion of T cell responses in the presence of excessive amounts of Ag.

	Introduction

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Tcell activation is generally mediated by professional APCs. An important property of professional APCs is the expression of costimulatory molecules such as the B7 family (1, 2). Two ligands for B7 have been characterized, CD28 and CTLA-4. While it has been shown that CD28 augments T cell activation and influences the induction of T cell anergy (3, 4, 5, 6, 7), the role of CTLA-4 has been more difficult to assess.

Initially, CTLA-4 was proposed to costimulate T cells similar to CD28 (8, 9). There is, however, accumulating evidence that CTLA-4 may instead be a negative regulator of T cells. Cross-linking of CTLA-4 on activated T cells effectively down-regulated T cell responses in vitro (10, 11, 12, 13), and in vivo blocking of CTLA-4 enhanced autoimmune disease (14). Mice rendered deficient for CTLA-4 expression by gene targeting die within a few weeks after birth. Most T cells in these mice are activated, and the animals suffer from a lymphoproliferative syndrome. In addition, activated T cells infiltrate the tissues, and large amounts of autospecific Abs are generated (15, 16). This phenotype of CTLA-4-deficient mice also indicates a down-regulatory function for CTLA-4.

Biochemical evidence further supports a direct inhibitory role of CTLA-4 during T cell responses. SHP-2 binds to CTLA-4, which dephosphorylates Shc. Shc binds and activates Grb2 and Sos, forming an important link between the TCR and the Ras pathway (17). Together, this evidence suggests that CTLA-4 plays a crucial role to actively inhibit T cell responses.

It is not known at what stage of the immune response CTLA-4 interferes with T cell responsiveness. The kinetics of CTLA-4 expression are reported to be slow. No CTLA-4 is found on the surface of naive T cells, and maximal expression is usually found a few days after stimulation (3, 10). It is, therefore, generally assumed that CTLA-4 exerts its effects late during immune responses. CTLA-4 may, therefore, be important for deletion of specific T cells after elimination of Ag as the infection is resolved. However, it has recently been shown that CTLA-4 accumulates at the T cell APC-contact site within a few hours after activation (18). This latter finding would, therefore, be compatible with an additional early down-regulatory role for CTLA-4.

This study analyzed the importance of CTLA-4 for activation of T cells during the early phase of the immune response and the elimination of T cells after clearance of the Ag during the late phase of the immune response. As a model Ag, lymphocytic choriomeningitis virus (LCMV)³ was used. LCMV induces a potent CTL response that is responsible for the elimination of the virus. LCMV-specific CTLs proliferate rapidly in the first week after infection and reach maximal frequencies around day 8. CTL frequencies decline rapidly thereafter, and up to 99% of the CTLs die after elimination of the Ag (19, 20). T cells in mice infected with a high dose of LCMV proliferate only transiently and are rapidly eliminated from the repertoire (T cell exhaustion). Such infected mice cannot eliminate the virus and become virus carriers (20).

To study the LCMV-specific CTL response in vitro and in vivo, CTLA-4-deficient mice were crossed with a transgenic mouse line expressing a TCR specific for LCMV (21). T cell activation and proliferation were studied in vitro after stimulation with agonist peptides. To determine whether CTLA-4 might exert a costimulatory activity similar to that of CD28 during stimulation with partial T cell agonists (22) or, alternatively, whether CTLA-4-deficient T cells may react to partial T cell agonists that failed to stimulate the proliferation of normal T cells, CTLA-4-deficient T cells were also stimulated with partial T cell agonists. In vivo activation, proliferation, and elimination of T cells were assessed following infection of mice with low and high doses of LCMV. Surprisingly, CTLA-4-deficient T cells were activated, proliferated, and deleted during the decline of the immune response, similar to control cells.

	Materials and Methods

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Mice and viruses

Transgenic mice expressing a TCR specific for peptide LCMV GP_33–41 presented in association with H-2D^b (21) and gene-targeted mice lacking expression of CTLA-4 (16) and RAG-2 (23) have been described previously. TCR-transgenic RAG-2^-/- CTLA-4^-/- (TCR⁺RAG^-CTLA-4^-) and control TCR-transgenic RAG-2^-/- CTLA-4^+/- or TCR-transgenic RAG-2^-/- CTLA-4^+/+ (TCR⁺RAG^-CTLA-4⁺) were used for all experiments. The LCMV isolate WE was provided by Dr. R. M. Zinkernagel (Zurich, Switzerland) and was grown on L cells at a low multiplicity of infection.

Peptides

Peptides were generated at the Amgen Institute (Boulder, CO) by a solid phase method using the F-moc/tBu-based protocol on an ABI-431 instrument (Applied Biosystems, Foster City, CA). The crude product was purified by HPLC. The following peptides were used: p33, KAVYNFATM; A4Y, KAVANFATM; A6F, KAVYNAATM; and MB6, KAVVNIATM; p33 defines the major CTL epitope on the LCMV GP in the H-2^b haplotype (24). For stability reasons, the C-terminal C has been replaced by an M (25).

Induction of LCMV-specific primary in vitro responses

Spleen cells (1 x 10⁵ cells/well) from naive RAG-2-deficient CTLA-4-deficient mice or from RAG-2-deficient CTLA-4 wild-type LCMV expressing a transgenic TCR specific for LCMV GP (p33) were stimulated in vitro with spleen cells (10⁵/well or as indicated) pulsed with a range of peptide concentrations in 96-well plates. Proliferation was assessed at the indicated time points by means of [³H]thymidine incorporation. *CTLA-4 expression was analyzed 3 days after stimulation using anti-CTLA-4 Ab coupled to phycoerythrin (PharMingen, San Diego, CA).

In vivo expansion and effector cell induction

Spleen cells from TCR⁺RAG^-CTLA-4⁺ and TCR⁺RAG^-CTLA-4^- mice (10⁶ cells) were adoptively transferred into normal C57BL/6 recipient mice. One hour later, mice were challenged with LCMV. At the indicated time points, spleen cells were harvested, and the presence of TCR-transgenic cells was assessed by flow cytometry (FCM) analysis for CD8 (phycoerythrin; PharMingen) and transgenic V2 expression (FITC; PharMingen).

To assess cytolytic effector function, spleen cells were tested in a ⁵¹Cr release assay, using peptide-pulsed EL-4 cells. To distinguish between endogenous T cells from the C57BL/6 recipient mice and transferred TCR-transgenic T cells, an additional peptide was included (MB6) that is recognized by the TCR-transgenic T cells, but not by the polyclonal C57BL/6 mouse-derived T cells (26) (see Fig. 3B).

View larger version (25K): [in this window] [in a new window]   FIGURE 3. Normal proliferation of TCR-transgenic RAG-2 x CTLA-4-deficient T cells after stimulation with the weak agonists A4Y and A6F. TCR+RAG-CTLA-4- (triangles) and control TCR+RAG-CTLA-4+ (circles) spleen cells were stimulated with various concentrations of A4Y and A6F, and proliferation was assessed 48, 72, or 96 h later. One representative experiment of two is shown. Spontaneous proliferation of T cells in the absence of Ag was <200 cpm.

	Results

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Initial studies had shown that it was difficult to induce LCMV-specific CTL responses in CTLA-4-deficient mice, probably because the majority of T cells from young mice became rapidly activated (15, 16). To examine Ag-negative T cell responses in the absence of CTLA-4, transgenic mice expressing a TCR specific for peptide p33 of LCMV GP (21) were crossed with CTLA-4-deficient mice (16).

CTLA-4-deficient mice develop a lymphoproliferative disease within a few weeks after birth. The cause of this generalized activation of lymphocytes is not known, although several possibilities exist, including activation of T cells by undefined mouse pathogens and commensals or by self-ligands. Because it was possible that TCR-transgenic CTLA-4-deficient T cells expressed a second endogenous TCR -chain (27) and therefore exhibited nontransgenic specificity, they may became activated in vivo in young animals similar to nontransgenic T cells. These activated T cells may interfere with the responses generated by nonactivated T cells exhibiting transgenic specificity. To avoid this problem, CTLA-4-deficient TCR-transgenic mice were crossed with RAG-2-deficient mice (23). Since no TCR gene rearrangement occurs in the absence of RAG-2, TCR-transgenic RAG-2-deficient mice have a homogeneous T cell population with exclusively transgene-encoded specificity (not shown).

The aim of this study was to analyze the importance of CTLA-4 during Ag-specific T cell responses. To ensure that CTLA-4 was actually expressed on activated TCR-transgenic T cells, we stimulated spleen cells from TCR⁺RAG^-CTLA-4⁺ and TCR⁺RAG^-CTLA-4^- mice with peptide p33-pulsed APCs and analyzed the expression of CTLA-4 3 days later by surface staining with anti-CTLA-4 Abs and subsequent FCM analysis (Fig. 1). CTLA-4 wild-type, but not CTLA-4-deficient, T cells expressed CTLA-4 after activation.

View larger version (14K): [in this window] [in a new window]   FIGURE 1. Induction of CTLA-4 surface expression after peptide-specific T cell activation. TCR+RAG-CTLA-4+ (left panel) and TCR+RAG-CTLA-4- (right panel) spleen cells were stimulated with p33 for 3 days, and surface expression of CTLA-4 was analyzed. Shaded areas represent nonstimulated spleen cells.

View larger version (20K): [in this window] [in a new window]   FIGURE 2. Normal proliferation of TCR-transgenic RAG-2 x CTLA-4-deficient T cells after stimulation with the strong agonist p33. TCR+RAG-CTLA-4- (triangles) and control TCR+RAG-CTLA-4+ (circles) spleen cells were stimulated with graded concentrations of p33, and proliferation was assessed 48, 72, or 96 h later. One representative experiment of two is shown. Spontaneous proliferation of T cells in the absence of Ag was <200 cpm.

We have recently shown that peptides exhibiting substitutions at position 4 or 6 of p33 may act as partial agonists or antagonists (22, 28, 29). Some of the partial agonist peptides were able to sensitize target cells for lysis, but failed to induce T cell proliferation (29). It is possible that CTLA-4-deficient T cells may be more reactive toward these ligands than are CTLA-4 wild-type T cells. Thus, to determine whether altered responses were detected in the absence of CTLA-4, T cells were stimulated with these peptides in the absence of CTLA-4. No alterations in the transgenic T cell response was detected with these altered peptide ligands (Fig. 3). Note that higher peptide concentrations were used for the altered peptide ligands than for p33 so as to be in the appropriate range of Ag concentrations to be able to potentially detect both increased and decreased proliferation of CTLA-4-deficient T cells. This result indicates that CTLA-4-deficient T cells are not hyper-reactive toward weak peptide ligands. Since we have previously shown that A4Y-specific proliferation is dependent on costimulation by CD28 (22), the experiment also suggests that CTLA-4 does not exhibit a similar role.

Normal in vivo induction of LCMV-specific T cell responses in the absence of CTLA-4

The in vitro experiments had suggested that T cell activation occurred normally in the absence of CTLA-4. It was, however, important to extend these studies in vivo. Due to the high precursor frequency of specific T cells in TCR-transgenic mice, T cell proliferation cannot be easily followed after viral infection. However, both expansion and elimination of transgenic T cells can be evaluated after adoptive transfer of transgenic T cells into normal recipient mice followed by infection with LCMV (20, 30). Spleen cells from control TCR⁺RAG^-CTLA-4⁺ and TCR⁺RAG^-CTLA-4^- (1 x 10⁶ cells) were transferred into normal, nonirradiated C57BL/6 mice that were subsequently immunized with a low dose of LCMV (200 plaque forming units (pfu)). This protocol has been shown to lead to rapid expansion of specific T cells that can be followed by FCM analysis of V2-expressing CD8⁺ T cells. Rejection of transferred cells due to minor histocompatibility complex differences does not occur until 2 wk after transfer (20, 30). Thus, it is possible to study the fate of transferred cells for at least 10 days. Both CTLA-4-deficient and CTLA-4 wild-type control T cells had strongly expanded 8 days after transfer (Fig. 4A). High lytic activity of spleen cells was observed on p33-coated EL-4 target cells (Fig. 4B). However, it was not possible to distinguish between the lytic response of transgenic T cells and the response of endogenous T cells of the recipient mice with this approach, since mice that had received normal, nontransgenic spleen cells also responded strongly in the ⁵¹Cr release assay (Fig. 4B).

View larger version (37K): [in this window] [in a new window]   FIGURE 4. Normal in vivo expansion and effector cell generation of RAG-2 x CTLA-4-deficient T cells. CTLA-4-deficient (CTLA-4 -/-) and control CTLA-4-wild-type (CTLA-4 +/+) TCR-transgenic RAG-2-deficient spleen cells (106 cells) were adoptively transferred into C57BL/6 recipient mice. One hour later, recipient mice were challenged with 2 x 102 pfu of LCMV. A, The presence of TCR-transgenic T cells was assessed 8 days after infection by staining for CD8 and V2. B, The presence of lytic effector cells was assessed 8 days after infection in a 51Cr release assay. EL-4 cells pulsed with p33 (closed triangles) or MB6 (open triangles) were used as target cells. Note that peptide MB6 is specifically recognized by the transgenic TCR but not by the polyclonal LCMV-specific T cells. Specific lysis of control EL-4 cells was <10%. One representative experiment of three is shown.

Following immunization of normal mice with low doses of LCMV, CTL precursor frequencies rapidly increase, peak around day 8, and decline thereafter to lower frequencies (19, 20). After infection with low virus doses, viral titers peak around day 4, while the virus is eliminated below the detection level by days 7 to 8 (20). Mice immunized with very high doses of LCMV may mount a transient, inefficient CTL response, and all CTL precursors are exhausted from the repertoire within a few days. Under these conditions, the virus is not eliminated, and a virus carrier status is established (20).

To assess the kinetics of CTL precursor frequencies in the absence of CTLA-4, the adoptive transfer experiments were repeated using low (100 pfu), intermediate (10⁴ pfu), and high (10⁶ pfu) doses of LCMV. The presence of transgenic T cells (Fig. 5A) and the lytic activity of the spleen cells (Fig. 5B) were assessed 4, 7 and 10 days later. As shown in Figure 5A, the kinetics of the expansion and deletion of transgenic T cells and of their lytic activity were identical in the presence and the absence of CTLA-4 for the high and intermediate virus doses. The expansion of transgenic T cells after low dose virus infection was also comparable in the presence and the absence of CTLA-4. Note that although CTLA-4-deficient T cells expanded somewhat more efficiently in this experiment, this was not consistently the case.

View larger version (25K): [in this window] [in a new window]   FIGURE 5. Normal expansion and elimination of RAG-2 x CTLA-4-deficient T cells. One million TCR+RAG-CTLA-4- (CTLA-4 -/-) and control TCR+RAG-CTLA-4+ (CTLA-4 +/+) were adoptively transferred into C57BL/6 recipient mice. One hour later, recipient mice were challenge with 102 (circles), 104 (squares), or 106 (triangles) pfu of LCMV. A, The presence of TCR-transgenic (TCR-tg) T cells (CD8+V2+) in spleens was assessed at the indicated time points by FCM analysis. B, The presence of effector cells was assessed in a 51Cr release assay using EL-4 cells pulsed with peptide MB6, which is specifically recognized by the TCR-transgenic T cells. Results are expressed as lytic units per spleen. Lytic units were determined for a threshold of 30% specific lysis. One representative experiment of two is shown.

Expansion of transferred T cells was low but clearly observable after infection with 10⁶ pfu of LCMV. This abortive immune response occurred in both the presence and the absence of CTLA-4 and is consistent with T cell exhaustion (20).

Thus, under the conditions tested, TCR-transgenic CD8⁺ T cells expanded normally in vivo and, after viral clearance around days 6 to 8 (not shown), were eliminated normally in the absence of CTLA-4. In addition, T cell exhaustion after infection with high virus doses did not critically depend on the presence of CTLA-4.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The present study analyzed the responsiveness of CD8⁺ cytotoxic T cells in the absence of CTLA-4. Surprisingly, no overt abnormality of CTLA-4-deficient T cells could be revealed. This is contrary to the general expectations and also does not readily fit the observation that CTLA-4-deficient mice die shortly after birth due to generalized activation of T cells. One explanation for this phenotype could be that CTLA-4-deficient T cells are more easily activated and are possibly hyper-reactive toward self-ligands. Alternatively, it is possible that CTLA-4 is involved in the down-regulation of an immune response and that CTLA-4-deficient T cells are not deleted following induction of an immune response. The results presented here argue against these possibilities and can be summarized as follows. 1) CTLA-4-deficient T cells efficiently proliferate and differentiate to lytic effector cells. 2) CTLA-4-deficient T cells do not proliferate for substantially longer time periods than control cells. 3) Activation of CTLA-4-deficient T cells does not require less ligand for T cell stimulation. 4) Partial agonists that fail to induce proliferation of normal T cells also fail to activate CTLA-4-deficient T cells. 5) Exhaustion of T cell responses by excessive amounts of Ag was not impaired in the absence of CTLA-4. Thus, the absence of CTLA-4 did not interfere with early or late aspects of T cell responses. In addition, other experiments indicate that thymic negative selection is normal in the absence of CTLA-4. This suggests that the phenotype of CTLA-4-deficient mice does not result from increased numbers of self-specific T cells (31). It, however, still remains possible that CTLA-4 may be involved in some aspect of peripheral tolerance induction. We show here that LCMV-specific T cells can be exhausted in the presence of excessive amounts of Ag in the absence of CTLA-4, arguing that at least peripheral deletion mechanisms are intact. It remains, however, possible that the induction of peripheral T cell anergy might be impaired. In fact, it has recently been shown that CTLA-4 could be important for the induction of T cell anergy (32).

The normal regulation of the immune response after adoptive transfer of CTLA-4-deficient T cells may also be controlled by undefined regulatory mechanisms, as, for example, by suppressive Th2 cells, that remain intact in the wild-type host. Alternatively, it may be possible that the generalized T cell activation is initially confined to CD4⁺ T cells, which subsequently activate CD8⁺ T cells. Thus, CTLA-4 may be more important for the fine tuning and/or termination of CD4⁺ T cell responses than for the CD8⁺ T cell responses, a phenomenon that has been described for induction of autoimmunity in the absence of IL-2 (33). In addition, CD8⁺ T cells may themselves directly interfere with their own responses by eliminating APCs. As a result, CD8⁺ T cells may be less dependent on down-regulatory mechanisms than are CD4⁺ T cells and are possibly solely regulated by the presence of Ag.

Finally, the absence of CTLA-4 may indirectly lead to the presence of abnormally activated T cells. Biochemical evidence suggests that CTLA-4 inhibits activation of the mitogen-activated protein kinase pathway early after stimulation of the TCR (17). It is, therefore, conceivable that CTLA-4-deficient T cells become hyperactivated rapidly after encounter of APCs because of an overwhelming signal 1 and subsequently hyperactivate the APCs. These abnormally activated APCs may then induce T cells that would not normally be stimulated, possibly leading to autoimmunity.

In summary, CTLA-4-deficient CD8⁺ T cells do not reveal gross abnormalities in their Ag-specific reactivity, indicating that CTLA-4 plays a minor role in regulating CD8⁺ T cell responses.

	Acknowledgments

 
We thank Arsen Zakarian for excellent technical assistance.

	Footnotes

 
¹ This work was supported by the Swiss National Science Foundation, the Medical Research Council of Canada, and a Medical Research Council scholarship (to P.S.P.).

² Address correspondence and reprint requests to Dr. Martin F. Bachmann, Basel Institute for Immunology, Grenzacherstr. 487, 4005 Basel, Switzerland.

³ Abbreviations used in this paper: LCMV, lymphocytic choriomeningitis virus; GP, glycoprotein; RAG-2, recombination-activating gene-2; FCM, flow cytometry; pfu, plaque-forming units.

Received for publication March 25, 1997. Accepted for publication September 18, 1997.

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