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[Cancer Research 61, 1107-1112, February 1, 2001]
© 2001 American Association for Cancer Research

Immunology

Optimal Induction of Effector but not Memory Antitumor Cytotoxic T Lymphocytes Involves Direct Antigen Presentation by the Tumor Cells1

Yannik Guilloux, Xue-feng Bai, Xingluo Liu, Pan Zheng and Yang Liu2

Department of Pathology and Comprehensive Cancer Center, Ohio State University Medical Center, Columbus, Ohio 43210 [X-f. B., X. L., P. Z., Y. L.], and Intituts de Biologie, INSERM U463, 44063, Nantes Cedex 1, France [Y. G.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
MHC class I-restricted tumor antigen can be presented to CD8+ T cells by two distinct mechanisms. Direct presentation involves degradation of cytosolic proteins by the proteosome into peptides, transport of the peptides across the endoplasmic reticulum membrane, and expression of the MHC-peptide complex on the tumor cell surface. Cross-presentation, on the other hand, involves uptake and intracellular processing of the tumor antigen by host antigen-presenting cells. Whereas it is clear that cross-presentation is necessary and sufficient for the induction of memory CTLs, it has not been tested whether such presentation is sufficient to induce effector CTLs. Here we analyzed the requirements of direct antigen presentation for the induction of effector and memory antitumor CTLs using a MHC class I- mutant incapable of direct antigen presentation and its parent, the MHC class I+ J558 cell line. We report that in comparison with the MHC class I+ tumor cell, the MHC class I- mutant induces equal priming for recall CTL response but poor effector CTLs. Our results demonstrate that optimal induction of effector CTLs, but not memory CTLs, requires direct antigen presentation by the tumor cells.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The host APCs3 play a critical role in the induction of immune response, including antitumor CTL response (1, 2, 3, 4) . Thus, studies with chimera mice suggest that the tumor antigens must be present on the host APCs to induce antitumor immunity (1 , 2) . More recently, we have demonstrated that B7-mediated antitumor CTL responses also require the presence of B7 on host APCs (5) . The requirement of both an antigen and a costimulator on host APCs raised an interesting issue with regard to the role of tumor cells in the induction of immunity. At least two hypotheses can be considered. First, tumor cells are merely donors of tumor antigen, and direct interaction between T cells and tumors is not involved in the induction of antitumor CTL response. Second, induction of antitumor CTL responses requires T-cell interaction with tumors as well as with host APCs.

Huang et al. (1) reported that tumor cells devoid of MHC class I induced a recall antitumor CTL response. This experiment offered strong support for the first hypothesis. In addition, experiments (1 , 2) using lethally irradiated mice that were reconstituted with bone marrow from one parent demonstrated that the CTL response was restricted by the MHC on bone marrow-derived cells. However, it was suggested recently that specific expression of the restricting MHC on bone marrow-derived dendritic cells may be required for survival of naïve T cells (6) . If this hypothesis is substantiated, the data derived from chimera mice may need to be reinterpreted.

The major difficulty in the first model is imposed by the fact that expression of B7 on the tumor cells significantly enhances antitumor immunity in numerous models (7, 8, 9, 10, 11) . These findings argue for a more active role of tumor cells in the induction of antitumor immunity. In this regard, a recent report suggested that B7-transfected tumor cells directly primed antitumor CTL response (12) .

A successful CD8 T-cell response has two major products: effector and memory T cells. Whereas these two populations of T cells are difficult to distinguish by the cell surface markers (13 , 14) , they have distinct immunological properties. First, effector T cells are cytolytic to tumors and virus-infected cells, whereas memory T cells generally have poor cytotoxicity without in vitro restimulation (15) . Second, memory T cells have a long life span in the absence of nominal antigens (16, 17, 18) , whereas the majority of the effector T cells undergo programmed cell death after clearance of antigens (19, 20, 21) . Third, our recent study revealed that costimulation by the heat-stable antigen leads to induction of memory T cells, but not effector T cells, which indicates that induction of memory and effector T cells can use distinct costimulatory molecules (22) . Finally, recombinant MHC consisting of ß2M, H-chain, and an antagonist peptide induces expansion and acquisition of memory markers but not the effector function of T cells in vivo (23) .

The distinction between memory and effector T cells may help to reconcile the apparent contradiction in the field. Because recall responses measure memory cells but not necessarily effector T cells (15) , one may suggest that direct interaction between T cells and tumors is required for the induction of effector cells but not memory cells. To test this hypothesis, we used a tumor model that allowed direct measurement of ex vivo CTL effector (5 , 9 , 10) . Moreover, we have recently produced a recurrent tumor line that lacks expression of cell surface MHC class I due to multiple antigen presentation defects (24) . To determine the requirement for direct antigen presentation by tumor cells in the induction of effector and memory cells, we produced plasmacytoma lacking MHC class I and/or costimulatory molecule B7-1. We compared the tumor variants with the parent tumor cell line for their ability to induce memory and effector antitumor CTLs. Here we report that optimal induction of effector but not memory CTLs requires direct antigen presentation by the tumor cells. This finding reveals an active role of tumor cells in the induction of antitumor CTLs.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Tumor Cell Lines and Mice.
All tumor cell lines used for in vivo study were derived from J558, a plasmacytoma of BALB/c origin. The J558 cell line expressing murine costimulatory molecule B7-1 has been described previously (9) . Because it expresses high levels of cell surface MHC class I, including Ld, it is henceforth called B7+Ld+. We have recently described a recurrent tumor cell line, ReB7, that lacks cell surface MHC class I due to multiple antigen presentation defects (24) . Here we isolated two subclones from the cell line by limiting dilution. One cell line, which expresses B7-1 but not MHC class I, is called B7+Ld-, whereas the other, which has lost both B7 and MHC class I, is called B7-Ld-. Proto-oncogene pml (10A7)- or vector (SG9)-transfected B7+Ld- cell lines, as described previously (24) , were also used.

Male BALB/c and C57BL/6j 6–18-week-old mice were purchased from the National Cancer Institute (Bethesda, MD). Transgenic mice expressing T-cell receptors specific for P1A have been described previously (25) .

Quantitation of Effector CTLs by Direct Cytotoxicity of ex Vivo TILs.
TILs isolated from tumors were used as effectors in a cytotoxicity assay without any in vitro restimulation, as described previously (9) . Data were presented in two forms. Specific lysis percentage was calculated as described previously (10) . The cytolytic unit was defined as a unit of activity that causes 50% specific lysis of 104 P388D1 cells pulsed with tumor antigenic peptide P1A. It was calculated according to the following formula: cytolytic units/106 TILs = (1/y) x 100; where y is the E:T at which 50% specific lysis of 104 target cells is achieved, as calculated by linear regression. R2 values range from 0.939 to 0.998. Whereas six dilutions were carried out for each effector, data from three to four dilutions that fell within the linear ranges were used.

Limiting Dilution Analysis.
To quantify the number of precursor cells, 24 replicas of given numbers of spleen cells were stimulated with 1 µg/ml P1A peptide in the presence of 1% supernatants from activated EL4 cells and 2 x 105 irradiated syngeneic feeder cells/well. After 7 days, the cultures were split into two and washed with medium. Five x 103 P1A-pulsed or control NP peptide-pulsed, 51Cr-labeled target cells were added to the wells to determine both the specificity and cytotoxicity of the microcultures. The wells with specific lysis equal to or greater than 3 SDs in medium release were scored as positive. The few wells that scored positive for both P1A and control NP peptides were excluded from the analysis. The numbers of precursors per 106 spleen cells are calculated based on precursor frequency.

In Vivo Priming of Alloreactive T Cells.
C57BL/6j mice received i.p. injection of 106 B7+Ld+ or B7+Ld- J558 cells. At different times after immunization, given numbers of spleen cells were stimulated with 104 irradiated (10,000 rads) B7+Ld+ J558 cells for 4 days. After washing, 51Cr-labeled mastocytoma P815 (H-2d) or EL4 (H-2b) cells were added, and the cytotoxicity of the spleen cells was measured in 6-h assays.

Flow Cytometry.
Cell surface expression of MHC class I Ld was determined using mAb HB27, whereas the cell surface expression of B7-1 was measured using anti-B7-1 mAb 10.16A.1, as described previously (26) .

Northern Blot Analysis of P1A mRNA Expression.
Total cytoplasmic RNA from different lines of J558 tumor cells was prepared and separated in agarose gel as described previously (10) . Radioactive probe was prepared as described previously (10) , except that the template used is a PCR product covering nucleotides 206–725 from the P1A open reading frame.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Tumor Variants Used to Address the Requirement of Direct versus Indirect Antigen Presentation in Vivo.
We have recently characterized a recurrent tumor cell line derived from J558-B7 called ReB7, which has lost expression of cell surface MHC class I and multiple antigen presentation genes (TAP-1, TAP-2, LMP-2, and LMP-7) due to a malfunction of proto-oncogene pml (24) . In the current study, we obtained a B7+Ld- subclone by limiting dilution. We removed the B7-1+ cells among the ReB7 cell line by fluorescence-activated cell sorting and obtained, by limiting dilution, a subclone devoid of both B7-1 and MHC class I, which we call B7-Ld-. The cell surface phenotypes of the four J558 variants are shown in Fig. 1Citation .



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  		Fig. 1. Phenotype of J558 variants used in the study. Four J558 variants were incubated with either medium control, anti-B7-1 mAb 10.16A.1, or anti-Ld mAb HB27. The amount of mAb bound to the cell surface was determined by FITC-conjugated second-step reagents. Goat antimouse IgG (G@mou) was used for anti-H-2 Ld mAb, whereas goat antihamster IgG (G@ham) was used for anti-B7 mAb.

 
We have reported that the major tumor antigen in the J558 tumor cell line is P1A (10) . Because P1A-specific CTL response was the primary readout of the experiments, we determined the expression of the P1A gene among different cell lines. As shown in Fig. 2Citation , the four lines of J558 cells expressed a comparable amount of P1A mRNA.



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  		Fig. 2. Comparable amount of tumor antigen P1A expression among the J558 cell lines used for the study, as measured by Northern blot of cytoplasmic RNA using a 32P-labeled P1A probe. The amount of RNA loaded into each lane is shown in the bottom panel.

 
A critical element of the study is that the MHC class I- cell lines used in the study remain MHC- in vivo. The MHC class I- variants were isolated from mice that have strong antitumor CTLs (28) , which strongly indicates that they did not express MHC in vivo. Because priming alloreactive T cells requires direct antigen presentation, we compared B7+Ld- and B7+Ld+ for their ability to prime H-2d-reactive CTLs in vivo. We titrated spleen cells from naïve and primed mice and stimulated them with B7+Ld+ cells in vitro. Due to the high frequency of alloreactive T cells, naïve spleen cells can produce alloreactive CTLs after 4 days of culture in vitro. At days 8, 10, and 12 after immunization with B7+Ld+ cell lines, substantially stronger alloreactive CTLs can be detected (Fig. 3Citation ; data not shown). The peak response was detected on day 10 (data not shown). However, at none of the time points tested did immunization with the B7+Ld- cell line enhance alloreactive T-cell responses (Fig. 3Citation ; data not shown). These results confirm that the B7+Ld- tumor cell line cannot present antigen to T cells directly.



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  		Fig. 3. B7+Ld- J558 variant cannot prime alloreactive T cells in vivo. C57BL6/j mice were immunized with 106 of either B7+Ld+ or B7+Ld-. On days 8, 10, and 12 after immunization, spleen cells were harvested. Given numbers of spleen responders were stimulated with B7+Ld+ J558 cells for 4 days, and CTL activity was determined using P815 (H-2d) or EL4 (H-2b) targets. Data shown were the means and SD of quadruple samples of naïve or day 10 primed spleen cells.

 
Distinct Requirement for Induction of Memory and Effector CTLs.
We have previously established that the major tumor antigen in J558 cells is P1A peptide 35–43 presented by Ld and that strong P1A-specific effector CTLs can be detected among the ex vivo TILs (5 , 9 , 27 , 28) . Moreover, the antigen-specific CD8 T cells did not differentiate into effectors until they reached the tumors (5) . To determine whether induction of effector T cells requires direct antigen presentation, we isolated tumor-infiltrating cells from B7+Ld+, B7-Ld+, B7+Ld- , and B7-Ld-tumors. As shown in Fig. 4Citation , at 3 weeks after tumor inoculation, TILs from B7+Ld+ tumors have strong cytotoxicity against P1A-pulsed target cells but not against control influenza nucleoprotein peptide-pulsed target cells. In contrast, TILs from B7+Ld- tumors have barely detectable cytotoxicity against P1A-pulsed target cells. Consistent with our previous observations (5) , B7-Ld+ tumors did not induce antitumor effector CTLs. As expected, tumors lacking both B7 and Ld failed to induce effector CTLs.



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  		Fig. 4. Induction of P1A-specific antitumor effector CTLs requires direct antigen presentation. TILs were isolated from tumors at 3 weeks after inoculation and used as effector for CTL assay. Target cells used were P388D1 (H-2d) pulsed with either tumor antigenic peptide P1A or influenza NP366-374 peptide.

 
We reported that expression of the proto-oncogene pml restored cell surface expression of MHC class I (23) . We inoculated pml- and vector-transfected B7+Ld- cells into syngeneic mice and analyzed whether the effector CTL response was restored by overexpression of PML. As shown in Fig. 5Citation , TILs in the pml transfectants are cytotoxic to both tumor cells and P1A peptide-pulsed target cells but not to unpulsed target cells. In contrast, vector-transfected B7+Ld- tumors induce no effector CTLs. The data in Figs. 4Citation and 5Citation indicate that optimal antitumor CTL response in vivo requires tumor presentation of both antigen and costimulatory molecules.



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  		Fig. 5. Proto-oncogene pml, which induced cell surface MHC class I, restored the ability of B7+Ld- cells to induce effector CTLs. pml (10A7)- or vector (SG9)-transfected cell lines were inoculated into BALB/c mice. TILs were isolated at 3 weeks. After removing natural killer cells with an anti-interleukin 2 receptor ß chain mAb, the TILs were tested for their cytotoxicity to P1A peptide-pulsed or unpulsed targets. a, expression of P1A in the 10A7 and SG9 cell lines, as determined by Northern blot. Top panel, P1A RNA; bottom panel, total RNA loading. b and c, CTL activity of TILs isolated from the 10A7 or SG9 cell line. The target cells used in b are B7+Ld+ J558 cells, whereas those used in c are P388D1 cells pulsed with (+P) or without (-P) P1A peptide.

 
A comparison of the data in Fig. 5Citation and Fig. 4Citation indicate that the pml transfectant 10A7 induced a poorer effector CTL response in vivo than did J558-B7. This is most likely due to the fact that 10A7 expressed less cell surface MHC than did J558-B7, as shown in Fig. 6aCitation . Moreover, 10A7 is less sensitive to lysis by P1 CTLs (Fig. 6b)Citation . The correlation between cell surface MHC levels and the strength of effector CTL response is consistent with a critical role for direct antigen presentation in the induction of effector CTLs.



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  		Fig. 6. Comparison of tumor cell lines for their cell surface MHC and susceptibility to P1CTL. a, H-2 Ld expression on B7+Ld- tumor cell lines transfected with either pml (10A7) or vector control (SG9) in comparison to the B7+Ld+ cell line J558-B7. Data presented are histograms of the three cell lines stained with either biotinylated HB27 followed by phycoerythorin-streptavidin (solid lines) or the second-step reagent alone (dotted lines). b, lysis of the cell lines by in vitro stimulated transgenic T cells. Data are from a 6-h CTL assay.

 
One potential interpretation of the lack of effector CTLs in the TILs in MHC class I- tumors is that the effectors are produced in the lymphoid tissues but fail to migrate to the tumor due to a lack of Ld-P1A complex on tumor cells. To test this, we tested the ex vivo cytotoxicity of spleen cells from mice that have borne the B7+Ld- tumors for 4 weeks. As shown in Fig. 7Citation , no ex vivo effector cells that could recognize P1A-pulsed target cells were detected. As a positive control, activated transgenic P1A-specific T cells (25) efficiently lysed the target cells.



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  		Fig. 7. Spleen cells from mice bearing B7+Ld- tumors do not have ex vivo P1A-specific CTLs. Spleen cells were isolated from the mice at about 4 weeks (Sp) after tumors became palpable and were used as effectors; P388D1 cells were labeled with 51Cr and used as targets. P1A antigen (P1A; 1 µg/ml) or medium control (ctrl) was cocultured with targets during the CTL assay. Spleen cells from transgenic mice (TG) expressing T cell receptor specific for P1A (25) were stimulated with P1A peptide (1 µg/ml) for 4 days and used as positive control.

 
The requirement of both MHC and costimulators on the tumor cells for CTL response in vivo appears contradictory to earlier observations that MHC class I- tumors can induce recall CTLs (29) . However, because previous studies evaluated recall CTL response rather than ex vivo effectors, one way to reconcile these observations is that induction of memory cells does not require direct antigen presentation from tumors. We therefore compared memory CTL response in the same four groups of mice used in Fig. 4Citation by limiting dilution.

Limiting dilution does not necessarily distinguish effector versus memory T cells. However, in the absence of effector cells, increased numbers of CTLps reflect the presence of memory T cells. No effector T cells were detectable in the TILs from the J558 B7+Ld-, B7-Ld+, and B7-Ld- tumors (Fig. 3)Citation , nor was the ex vivo effector detectable in the spleen of mice bearing B7+Ld- tumors (Fig. 4)Citation . With regard to J558 B7+Ld+, although the effector CTLs were detected in the tumors, we have not observed any CTL effectors in the spleen (5) . It is therefore most likely that increased CTLps in the spleens from all four groups of mice reflect memory response. As shown in Fig. 8Citation , there is no significant difference among mice that received injections of J558-B7+Ld+, J558-B7+ Ld-, J558-B7-Ld+ or J558-B7-Ld-. Thus, Ld expressed on tumors seems to be unessential for memory response. In contrast, a comparison between CTLps in spleens from mice injected with J558 B7+Ld+ and J558-B7- Ld+ or J558-B7+Ld- and J558-B7- Ld- reveals that expression of B7 increases precursor frequency.



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  		Fig. 8. Limiting dilution analysis to determine the precursor frequency of P1A-specific CTLs. Grading numbers (24 replicates/dose) of spleen cells from naïve or tumor-bearing mice used in the experiment in Fig. 4Citation were used as responders. The responder cells were stimulated with p1A peptides and irradiated spleen cells as feeders for 7 days in 96-well plates. The cultures were split into two and tested for cytotoxicity in P1A-pulsed or control NP peptide-pulsed targets. The precursor frequency is presented in the legends. N.D., not determined.

 
A comparison between memory and effector CTL responses at different time points after tumor challenge is presented in Table 1Citation . These results reveal two interesting points. First, whereas optimal effector CTL response depends on MHC class I expression on the tumor, no such requirement is observed for the induction of memory CTL response. On a cell-to-cell basis, TILs from B7+Ld+ tumors have 4–5-fold more effector T cells. Because 2–3-fold more TILs were obtained from B7+Ld+ tumors than from B7+Ld- tumors (data not shown), B7+Ld+ tumors of similar mass may be infiltrated with 10–15-fold more effector cells than the B7+Ld- tumors. Second, induction of memory and effector T cells follows different kinetics. On day 11 after tumor inoculation, when effector CTLs are barely detectable, memory response is already at its peak. These results illustrate that distinct mechanisms are involved in the induction of memory and effector CTL responses.


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  		Table 1 Requirement for direct antigen presentation for optimal induction of effector but not memory cells

TILs isolated from 2–3 tumors/group were used as effectors for CTL assay. The CTL data were processed by linear regression analysis and presented in cytolytic units (see "Materials and Methods"). Spleen cells were harvested from the same tumor-bearing mice to determine CTLp.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
An antitumor CTL response involves production of both memory and effector T cells. The former is responsible for the recall responses, whereas the latter is responsible for specific destruction of tumor cells. Perhaps because of the intrinsically poor immunogenicity of tumors, it has been difficult to detect tumor-specific CTLs without in vitro restimulation. In the absence of effector T cells, recall responses are functions of memory cells. It is therefore likely that much of our knowledge about the induction of antitumor CTL responses may have been derived from experiments that use immune memory as their final readouts. Because effector cells are immunologically distinct from memory cells, an interesting issue is whether the induction of effector T cells is governed by similar principles.

It has been postulated that antitumor CTL responses are induced by the host APCs that uptake and cross-present tumor antigens (1 , 2) . Whereas this concept explains the effective induction of antitumor CTL memory response by MHC class I- tumor cells (1) , it does not predict significant enhancement of antitumor immunity by B7 on tumor cells (7, 8, 9, 10, 11 , 30) . This apparent contradiction can be explained if induction of effector but not memory T cells requires direct antigen presentation by the tumor cells (Fig. 9)Citation . The results presented in the current study provide strong support for the new model. Because these data are obtained from the J558 model, it remains to be determined whether or not the conclusion can be generalized into other tumor models.



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  		Fig. 9. A schematic model to explain distinct mechanisms and sites of antitumor memory and effector CTL responses. Naïve CD8 T cells are stimulated in lymphoid tissues, such as local draining lymph nodes, by host APCs that migrated from the tumor, where the tumor antigens are captured. Such stimulation leads to the production of memory T cells and activated T cells. The activated T cells migrate into the tumor and differentiate into effector T cells when they receive an antigenic stimulation by the tumor. Alternatively, activated T cells may stay in the lymphoid tissue and differentiate into memory T cells. The memory T cells may migrate into tumors to differentiate into effectors on tumor stimulation.

 
We have analyzed the requirement for tumor expression of MHC class I and costimulatory molecules using tumor variants that have lost MHC and/or costimulatory molecules. Using limiting dilution to measure precursors for tumor antigen P1A-specific CTLps in spleens devoid of effector CTLs, we confirmed the previous observations that induction of memory cells does not require direct antigen presentation (1 , 2 , 31) . Interestingly, we found that optimal induction of effector T cells in the tumors requires direct antigen presentation by the tumors. This is consistent with the recent finding in MHC class II-restricted antitumor immunity (32) .

We have avoided using the irradiation bone marrow chimera model to address the essential function of host APCs for two reasons. First, a recent study demonstrated that survival of naïve T cells may require the restricting MHC element of the host dendritic cells (6) , which would make it impossible to determine whether the host APCs were required for survival or priming of naïve T cells. Second, we have already reported in this model that activation of tumor-specific T cells requires costimulation by host APCs by a different approach (5) . As such, our model would predict lack of effector cells in the absence of host APCs (see below).

The immunological basis for the requirement for direct antigenic stimulation in the induction of effector CTLs is still unclear. A simple interpretation is that antigenic density generated by cross-presentation may be too low to induce effector cells, as we have recently suggested (5) . We have shown that induction of effector T cells also requires costimulation by B7-1/B7-2 on both tumors and host APCs (5) . It is therefore most likely that induction of effector CTLs requires both direct presentation and cross-presentation of antigen, perhaps in a stepwise fashion (Fig. 9)Citation . Moreover, the tumor cells used in the current study are not metastatic because they do not migrate into local lymph nodes (5) . Because the traffic of naïve lymphocytes is primarily restricted to the blood and the lymphoid system, it is most likely that T cells for the tumor antigen first encounter antigens on host APCs in the lymphoid tissue. We suggest that this interaction leads to the production of memory cells and activated T cells that are not yet effector T cells. The activated T cells then migrate into the tumor and interact with the tumor cells directly. It is after this direct T-cell-tumor interaction that the mature effector CTLs are produced. This stepwise model explains priming by MHC class I- tumor cells as well as enhancement of antitumor immunity by B7 and MHC class I expressed on the tumor cells.


   ACKNOWLEDGMENTS
 
We thank Dr. S. Rath for helpful discussions, Dr. John Hirst for assistance in flow cytometry, Dr. Marc Bonneville for critical reading of the manuscript, and Jennifer Kiel for secretarial assistance.


   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 Supported by NIH Grants AI32981 and CA58033, New York University Medical Center Kaplan Comprehensive Cancer Center, and Ohio State University Comprehensive Cancer Center. Back

2 To whom requests for reprints should be addressed, at Department of Pathology, Ohio State University Medical Center, 129 Hamilton Hall, 1645 Neil Avenue, Columbus, OH 43210. Phone: (614) 292-3054; Fax: (614) 688-8152; E-mail: liu-3{at}medctr.osu.edu Back

3 The abbreviations used are: APC, antigen-presenting cell; TIL, tumor-infiltrating lymphocyte; mAb, monoclonal antibody; CTLp, CTL precursor; ER, endoplasmic reticulum; NP, nucleoprotein; TCR, T cell receptor; PE, phycoerythorin. Back

Received 12/ 2/99. Accepted 11/20/00.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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