TCR Vß usage and clonality of T cells isolated from progressing and rejected tumor sites before and after in vitro culture

Robert A. Kurt, Julie A. Park1, Samuel F. Schluter1, John J. Marchalonis1 and Emmanuel T. Akporiaye1

Earle A. Chiles Cancer Research Institute, 4805 NE Glisan Suite 5F40, Portland, OR 97213, USA
1 Department of Microbiology and Immunology, University of Arizona, Tucson, AZ 85724, USA

Correspondence to: R. A. Kurt


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
A gelatin sponge model of concomitant tumor immunity was employed in order to examine the clonality of T cells associated with progressing and rejected tumor sites. Here we show that freshly isolated T cells bearing TCR Vß1, CDR3 RPGTGN, Jß1.1 and TCR Vß8, CDR3 GD, Jß1.6 predominated progressing and rejected tumor sites. Despite the similarity in T cell populations, the T cells from rejected tumor sites were capable of killing the autologous tumor cells, whereas T cells from progressing tumor sites were not able to do so. The differing cytolytic ability could not be attributed to a difference in TCR {zeta} chain protein expression levels between both T cell populations. After a 5 day mixed lymphocyte tumor culture the T cells from the progressing tumor site were capable of killing autologous tumor cells, which suggested changes took place within the cell population during in vitro culture. Further TCR analysis revealed T cells bearing TCR Vß1, CDR3 RPGTGN, Jß1.1 and TCR Vß8, CDR3 GD, Jß1.6 were not expanded following the in vitro culture. These data suggest that the lack of cytotoxicity of freshly isolated tumor-infiltrating lymphocytes (TIL) was not due to abnormal TCR {zeta} chain expression or major differences in the TCR Vß usage. Additionally, the gain of TIL effector function did not correlate with an expansion of the TCR bearing T cells found to predominate the in vivo response. These data suggest that the predominant TCR Vß used by lymphocytes infiltrating regressing or rejected tumors may not represent the tumor reactive T cells that grow in culture or respond to the autologous tumor in vitro.

Keywords: T lymphocyte, TCR {zeta}, tumor immunity, tumor-infiltrating lymphocyte, tumor-rejecting lymphocytes


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The absence of palpable tumors following tumor rejection has made it virtually impossible to study the phenotypic and functional characteristics of tumor-infiltrating lymphocytes (TIL) during tumor rejection. Until recently, melanomas undergoing spontaneous regression have provided the best model for studying tumor rejection (1,2). We have employed a gelatin sponge model of concomitant tumor immunity in order to capture T cells associated with rejection of a moderately immunogenic murine mammary carcinoma, EMT6 (3,4). This model allowed a simultaneous comparison of T lymphocytes infiltrating progressing tumors and tumors undergoing rejection in the same host. We demonstrated that whereas TIL from the progressing tumor were not cytolytic, the T cells from the rejection site [tumor-rejecting lymphocytes (TRL)] mediated cytolysis of autologous tumor cells in vitro (35). Previously we reported a similarity in the TCR Vß usage between the freshly isolated TIL and TRL, and showed IFN-{gamma}, tumor necrosis factor-{alpha} and IL-10 were present at greater levels in rejected tumor sites, and transforming growth factor-ß was present at greater levels in progressing tumor sites by RT-PCR (5). In this report we examined TIL and TRL for differences in TCR {zeta} chain levels and the clonality of TCR Vß usage of freshly harvested and cultured tumor-associated lymphocytes.

Recently, several laboratories have reported alterations in the protein expression levels of molecules associated with TCR signaling during progressive tumor growth (69). Specifically, the TCR {zeta} chain has been found to be expressed at decreased levels in TIL from several human tumors (7,8) and animal models (69). Here we report that TIL and TRL showed similar protein expression levels of TCR {zeta}, and therefore the lack of cytolytic activity of freshly harvested TIL could not be attributed to decreased TCR {zeta} chain protein expression. However, TIL acquired effector function after incubation in an immunostimulatory environment suggesting changes took place during the in vitro culture.

The most variable region of the TCR ß chain, the CDR3, is involved in antigen binding (10). An examination of the CDR3 regions in TIL has been performed from several human tumors (11). These data demonstrate the clonality of T cells infiltrating progressing tumors and indicate the cells most likely involved in the anti-tumor immune response. In order to reveal the clonality of T cells infiltrating progressing and rejected EMT6 tumors we sequenced the CDR3 regions of TIL and TRL. An examination of the freshly isolated TIL and TRL revealed that TCR Vß1, CDR3 RPGTGN, Jß1.1 and TCR Vß8, CDR3 GD, Jß1.6 predominated both progressing and rejected tumor sites. Despite the presence of similar T cell populations, the TIL lacked tumoricidal activity. In an attempt to expand this population and generate cytotoxic T cells from the TIL population a mixed lymphocyte tumor culture (MLTC) was used. Following the MLTC, the TIL gained cytolytic activity and the TRL showed an increase in cytolytic activity toward the autologous tumor. However, the increase in cytolytic activity in both populations did not correlate with an expansion of the TCR Vß 1, CDR3 RPGTGN, Jß1.1 and TCR Vß8, CDR3 GD, Jß1.1 bearing T cells. These data suggest that the gain in TIL effector function and the increase in TRL effector function in vitro did not correlate with an expansion of the T cells found to predominate the in vivo anti-tumor response.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Mice
Female BALB/c mice (6–8 weeks old) were purchased from the Jackson Laboratory (Bar Harbor, ME). The mice were housed in the University of Arizona animal facility and fed ad libitum.

Tumor injection and sponge implantation
The EMT6 tumor cell line used in this study was kindly provided by Dr Sara Rockwell (New Haven, CT) and was maintained in culture as previously described (3). Mice were injected s.c. on the right flank with 1x105 viable EMT6 cells in 0.1 ml sterile PBS. Eight days later, tumor-bearing animals were anesthetized with pentobarbital (60 mg/kg body wt), and two 10 mmx10 mmx10 mm sterile gelatin sponges (Upjohn, Kalamazoo, MI) were implanted s.c. in the upper and lower dorsal regions. Separate incisions were made at each implant site to ensure sponge separation throughout the experiment. Two days following implantation (day 10), the sponges were injected with 1x105 EMT6 tumor cells. This injection schedule was based on our previous published reports (3,4) that demonstrated the acquisition of concomitant tumor immunity using a challenge time interval of 10 days between the primary and secondary tumor inoculations. By day 17, no tumor cells can be detected in the sponges, indicating a successful anti-tumor immune response was initiated.

Isolation of TIL and TRL
On day 17 after injection of the primary tumor, animals were killed by cervical dislocation. Sponges and tumors were removed, diced and digested in a collagenase cocktail as previously described (3). The resulting cell suspensions were filtered through a 140 µm wire screen and washed with {alpha}-MEM buffered with 20 mM morpholinopropane sulfonic acid and supplemented with 10% FBS ({alpha}-10-MOPS).

T lymphocytes were highly enriched from progressing tumors and sponges using the MiniMACS microbeads system (Miltenyi Biotec, Auburn, CA). Tumors and sponges, digested as previously described (3), were depleted of macrophages by plastic adherence and then passed over a nylon wool column to remove additional macrophages and B cells. Approximately 1x106 cells were then labeled with 1 µg of rat anti-mouse CD11b (Caltag, San Francisco, CA) and 1 µg of rat anti-mouse Gr-1 (PharMingen, San Diego, CA), washed 1xin PBS, and resuspended in 80 µl of PBS. Goat anti-rat magnetic microbeads (20 µl, Miltenyi Biotec) were added and incubated at 4°C for 15 min undisturbed. The cells were washed 2xin PBS and resuspended in 500 µl PBS. The magnetic separation column (Miltenyi Biotec) was prepared by running 500 µl of PBS through it. The labeled cells were then pipetted into the column and allowed to run through. The eluent was collected and used as enriched T cells. TRL were 90% Thy1.2+ and TIL were 70% Thy1.2+ as determined by flow cytometric analysis.

MLTC
The T lymphocytes from progressing tumors (TIL) and sponges (TRL) were stimulated in vitro in a MLTC. The TIL and TRL were enriched as described above, and resuspended in {alpha}-RPMI 1640 supplemented with 10% heat-inactivated FBS, 5.5x10–5 M 2-mercaptoethanol, and 1.1x10–4 M sodium pyruvate. Approximately 1x106 cells were added to a single well in a 12-well plate (Falcon 3034; Becton Dickinson, Lincoln Park, NJ). Irradiated (65–70 Gy) EMT6 tumor cells were added at a concentration of 1x104 cells/well and irradiated (20 Gy) splenocytes (1x106 cell/well) were added as antigen-presenting cells. IL-2 (Amgen, Thousand Oaks, CA) was added at a concentration of 40 U/ml in a final volume of 5 ml/well. The low-dose IL-2 was sufficient for T cell survival, but not for LAK cell generation. The MLTC was then incubated, undisturbed, for 5 days at 37°C in 7% CO2. Non-adherent cells were harvested by vigorous pipeting.

Cytotoxicity assay
The cytotoxicity of autologous tumor cells by enriched TIL and TRL was determined in an 8 h 51Cr-release assay as previously described (3). Lymphocytes in both TIL and TRL populations were equalized based on lymphocyte content after Wright–Giemsa staining. Experiments were performed in triplicate and spontaneous release never exceeded 31%.

Vß TCR analysis
Because antibodies were not available for all of the Vß TCR we used RT-PCR to examine Vß TCR usage. In order to analyze the TCR Vß repertoire of TIL and TRL, progressing tumors and sponges were digested with collagenase as described above. Approximately 5x106 to 1x107 cells from three separate sponge and tumor digests were washed 2xin PBS, and poly(A) mRNA and total RNA were isolated using the Invitrogen (San Diego, CA) Micro Fast Track mRNA isolation kit and the Trizol reagent (Life Technologies, Grand Island, NY) respectively. Isolated RNA was routinely treated with DNase (Boehringer Mannheim, Indianapolis, IN) prior to cDNA synthesis. Analysis by PCR, of DNase-treated RNA, did not yield any product (data not shown) confirming an absence of DNA contamination. The resulting RNA was quantified by spectrophotometry and equal amounts of RNA from each source were reverse transcribed using Invitrogen's cDNA Cycle kit. Typically 10–100 ng mRNA or 5 µg total RNA was used per cDNA reaction. The cDNA was then submitted to semi-quantitative PCR using a panel of specific Vß 5' (sense) primers and a common Cß 3' (anti-sense) primer designed using the Oligo Program (National Biosciences, Plymouth, MN) as previously described (5).

CDR3 region analysis
In order to determine the clonality of TIL and TRL, the PCR products of the Vß which predominated each tumor site were ligated into the PGEM T vector (Promega, Madison, WI) and used to transform DH5{alpha} Escherichia coli strain (Stratagene Cloning Systems, La Jolla, CA). Ampicillin-resistant, white colonies were screened for insert by ApaI–PstI digestion. Plasmid DNA was then extracted and sequenced using the Sequenase version 2.0 DNA sequencing kit (US Biochemical, Cleveland, OH). The joining region of the TCR ß chain (Jß) was identified by comparison of these sequences to published Jß sequences (1214).

Western blotting
In order to investigate potential differences in the signaling machinery of the TIL and TRL, Western blots were performed in order to examine TCR {zeta}. For these studies the TIL and TRL were purified as described above and equalized for viability by acridine orange/propidium iodine staining followed by staining with the Leukostat staining kit (Fisher Diagnostics, Orangeburg, NY) to determine the percentage of lymphocytes in the samples. Approximately 1x106 viable lymphocytes were washed 2xin PBS containing 1 mM Na3VO4 (Sigma, St Louis, MO) and lysed in 0.15 ml cold lysis buffer (20 mM Tris–HCl, pH 8.0, 137 mM NaCl, 10% glycerol, 1% Triton X-100, 1 mM NaVO4, 2 mM EDTA, 1 mM PMSF, 1 µg/ml leupeptin, 1 µg/ml aprotinin and 1 µg/ml pepstatin) for 10 min on ice. The lysates were then centrifuged at 10,000 g for 10 min at 4 °C. The supernatants were transferred to clean 1.5 ml tubes and an equal volume of 2xSDS–PAGE buffer (18 ml dH2O, 0.12 g Tris base, 1 g SDS, 0.2 ml 0.5 M EDTA, pH 8.0, 0.5 ml 10% sodium azide, 0.1g bromphenol blue, 5 ml 2-mercaptoethanol and 25 ml glycerol) was added. The samples were stored at –20°C. For protein analysis, the lysates were heated to 95 °C for 5 min and separated on a 10% SDS–PAGE gel. The protein was transferred to a nylon membrane (Immobilon P; Millipore, Bedford, MA) at 80 V overnight at 4°C. The membranes were washed 3xfor 15 min each in 10 ml of blocking buffer [0.05% Tween 20/1% porcine gelatin type A (Sigma) in TBS] while rocking (Red Rocker; Hoeffer Scientific Instruments, San Francisco, CA). For TCR {zeta} analysis, the membranes were incubated with 1 µg/ml of anti-mouse CD3 {zeta} antibody (PharMingen) in 10 ml blocking buffer for 2 h while rocking. For CD3{varepsilon} analysis, the membranes were incubated with 1 µg/ml of anti-CD3{varepsilon} antibody (Dako, Carpinteria, CA) in 10 ml blocking buffer for 2 h while rocking. The membranes were washed in blocking buffer 2xeach for 5 min and incubated with a 1/6000 dilution of goat anti-mouse (TCR {zeta}) or anti-rabbit (CD3{varepsilon}) horseradish peroxidase conjugate (Sigma) for 45 min. The membranes were washed with TBS 3 times for 5 min each and developed by ECL according to the manufacturer's instructions (ECL Western blotting detection reagent; Amersham Life Science, Arlington Heights, IL).


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Cytolytic activity of TIL and TRL
In order to determine if T lymphocytes present in progressing tumors were capable of mediating cytolytic function against autologous tumor cells in vitro, they were re-stimulated in a MLTC. Although unable to lyse tumor cells when initially isolated, after 5 days stimulation in vitro, TIL were capable of lysing autologous tumor cells in an 8 h 51Cr-release assay (Fig. 1Go). Previous published work has shown that the cytolytic activity was not due to NK cell activity (5). After the MLTC, TIL exhibited 25% cytotoxicity at an E:T of 40:1. TRL were cytotoxic towards EMT6 tumor cells prior to (52%) and after (69%) in vitro stimulation (Fig. 1Go). These data indicated that the cytotoxic function of the TIL could be rescued after in vitro culture.



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Fig. 1. Cytotoxicity of TIL and TRL before and after MLTC. The ability of TIL and TRL to lyse EMT6 tumor cells after a 5 day MLTC was tested in an 8 h 51Cr-release assay. The error bars indicate the mean ± SD of triplicate samples. The data are representative of two separate experiments.

 
TCR {zeta} chain expression
In order to determine if the lack of cytotoxicity by freshly isolated TIL was associated with a decrease in the TCR {zeta} chain expression we compared TCR {zeta} chain protein levels in the freshly isolated TIL and TRL (Fig. 2Go). Western blot analysis revealed that the protein expression levels of TCR {zeta} were similar between the TIL and TRL (Fig. 2Go). These data suggested that the lack of TIL-mediated killing was not related to abnormal TCR {zeta} chain expression.



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Fig. 2. Western blot analysis of TCR {zeta} in TIL and TRL. In order to determine if there were any differences in the signaling abilities of the TIL and TRL, Western blots were performed using antibodies specific for TCR {zeta}. The amount of protein per lane is equivalent to 1.5x105 cells. Membrane B was probed with anti-TCR {zeta} antibody as indicated in Methods. Membrane A is the CD3{varepsilon} control for protein loading.

 
TCR analysis from freshly isolated cells
Since the differences in cytotoxic activities of TIL and TRL could be due to the presence of different T cell phenotypes, these effectors were examined for differences in their TCR Vß family usage by semi-quantitative PCR. The data revealed a similarity in the TCR Vß usage by TIL and TRL. The usage of TCR Vß1, 8, 10, 11 and 13 was increased in both populations compared to the remaining Vß (Fig. 3AGo). Although the level of usage of TCR Vß4, 5.2 and 7 was slightly higher in the TRL than the TIL, these values were not significantly different from their counterparts in spleens from naive non-tumor bearing animals (data not shown). The predominance of TCR Vß1 and 8 bearing T cells in TIL and TRL (Fig. 3AGo) suggested that these T cell populations may be involved in the in vivo antitumor immune response.



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Fig. 3. TCR Vß analysis of TIL and TRL before and after a 5 day MLTC. The TCR Vß repertoires of TIL and TRL were examined by RT-PCR before (A) and after (B) a 5 day MLTC. The PCR was performed using TCR Vß-specific primers and a common Cß primer. The data represent the TCR Vß usage from an average of six separate animals prior to MLTC and 10 animals after MLTC.

 
Although TCR Vß analysis of the TIL and TRL populations suggested that the same Vß families were responding to the progressing and rejected tumors, the Vß families may have differed within the antigen binding CDR3 region (10). In order to determine the clonality of the TCR Vß, the CDR3 regions of the most predominant TCR Vß in the TIL and TRL were sequenced (Table 1Go). In the Vß1 compartment of the progressing and rejected tumors, four of 10 and six of 10 TCRs respectively were Jß1.1 (Table 1Go). Three out of the 10 clones sequenced possessed identical diversity regions, CDR3 RPGTGN, associated with Jß1.1 in the progressing tumors and in the rejected tumors (Table 1Go). In the TCR Vß8 compartment, Jß1.6 was utilized in four of 10 TCRs from progressing tumors and in three of 10 TCRs from rejected tumors. The CDR3 sequence of these clones were identical and consisted of a glycine (G) and an aspartic acid (D).


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Table 1. CDR3 analysis of freshly isolated TIL and TRL
 
Significantly, the Vß1 TCR clones which predominated the EMT6 TIL and TRL (CDR3 RPGTGN) were not detected in the spleens of EMT6 tumor bearing animals despite an expansion of the Vß1 TCR bearing T cells in these spleens compared to the spleens of naïve animals (Table 2Go). Similarly, the Vß8 TCR clones which predominated the EMT6 TIL and TRL (CDR3 GD) were not detected in the spleens of EMT6 tumor bearing animals despite an expansion of the Vß8 TCR bearing T cells in these spleens compared to the spleens of naïve animals (Table 2Go). Moreover, the recruitment or expansion of these T cells appeared to be tumor dependent. There was no expansion of the Vß 1 TCR bearing T cells in a syngeneic mammary tumor system, 168 (Table 2Go). As in the EMT6 model, the Vß8 TCR bearing T cells were expanded in the spleens (9%), the TIL (36%) and TRL (68%) compared to the naive spleens (7%) in the 168 tumor system (Table 2Go). However, two unique clones predominated the Vß8 TCR bearing T cells in the 168 tumor system (CDR3 SPGR and SEWGGAG) which were not detected in the EMT6 system. Conversely the Vß8 TCR bearing clones which predominated the EMT6 TIL and TRL (CDR3 GD) were not detected in the Vß8 TCR bearing clones in the 168 system (Table 2Go). These data emphasize that the TCR bearing T cells recruited to the tumor sites were tumor dependent, clonal in nature and likely involved in the in vivo anti-tumor immune response.


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Table 2. Tumor-dependent recruitment of T cells to tumor sites.
 
TCR analysis after in vitro culture
The TCR Vß repertoires of TIL and TRL were analyzed after in vitro stimulation in order to determine if there was a preferential expansion of TCR Vß1 and 8 bearing T cells. The data instead revealed a spreading of TCR Vß usage during MLTC. Whereas a decrease in TCR Vß1 and 8 usage occurred, an increase in TCR Vß4, 5.1, 5.2, 6, 9, 12 and 15 usage was observed (Fig. 3BGo). Before in vitro expansion, T cells bearing TCR Vß1 represented 20% of the TIL and 19% of the TRL, and T cells bearing the TCR Vß8 represented 19% of the TIL and 14% of the TRL (Fig. 3AGo). After MLTC, the percent cells bearing TCR Vß1 decreased to 7% in the TIL and 6% in the TRL (Fig. 3BGo). Similarly, T cells expressing TCR Vß8 dropped to 12% in the TIL and 9% in the TRL (Fig. 3BGo). These data suggested that cells expressing TCR other than TCR Vß1 and 8 were expanded during in vitro stimulation and may be involved in antigen recognition in vitro.

One possible explanation for the decrease in Vß1 and 8 TCR-bearing cells after MLTC is that some clones expressing these receptors did not respond to the EMT6 tumor-associated antigens and died, leaving a population of clonally restricted cells. In order to determine whether this occurred, the CDR3 regions of the TCR Vß1 and 8 bearing T cells were sequenced after MLTC. The CDR3 region analysis of the TCR Vß 1 TIL showed there was an expansion in the usage of Jß1.1 from 40% before the MLTC to 80% following MLTC. Interestingly, the CDR3 sequence (RPGTGN) present in clones analyzed before the MLTC was absent in those sequenced post-MLTC (Table 3Go). Overall, three TCR Vß 1 TIL clones (CDR3; SPGQGN, SPGTGN and SQGLGDS) were shared between the TIL and TRL populations after the MLTC, whereas only 1 TCR Vß 1 clone (CDR3; RPGTG) was shared between the TIL and TRL populations before the MLTC. These findings suggest an increased clonality in the TCR Vß 1 TCR compartment during in vitro stimulation. In the TCR Vß8 compartment, Jß1.6 usage predominated both TIL and TRL clones in vivo (Table 1Go) but was absent in cells analyzed after in vitro stimulation (Table 3Go).


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Table 3. CDR3 analysis of the TIL and TRL after in vitro culture
 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Most investigations of antitumor immune responses focus on progressing tumors (1519). Tumor-associated lymphocytes from rejected or regressing tumors should provide information on the requirements for an effective antitumor immune response. To date only two studies have examined TIL in a spontaneously rejected human melanoma (1,2). In these studies, the TCR Vß usage and clonality of the T cell response was investigated. Whereas TCR Vß 16, CDR3 LRDSWN, Jß 2.1 was found to be the most predominant TCR used during the in vivo antitumor immune response (1), TCR Vß13, CDR3 WGGD, Jß1.1 was found to predominate a TIL line and clone grown from the bulk TIL population (2). These data suggest that the predominant TCR bearing T cells in vivo may not be the ones that respond to the tumor in vitro.

Our well-described model of concomitant tumor immunity (35) allows the simultaneous analyses of TCRs involved in tumor progression and rejection. Using this model we have shown that T cells isolated from progressing tumors were non-cytolytic, whereas T cells from rejected tumor sites were cytolytic towards the autologous tumor in vitro (35).

The lack of cytotoxicity by freshly isolated TIL suggested that either these cells were impaired or consisted of a T cell population different from TRL. In order to address the first possibility we examined TCR {zeta} chain expression. Abnormal TCR {zeta} chain expression in TIL from melanoma, colon cancer and renal cell carcinoma has been implicated as a factor contributing to progressive tumor growth (69). It has been alluded to in these reports that the abnormal TCR {zeta} expression may be related to the progressive tumor growth. The gelatin sponge model of concomitant tumor immunity provided a system to investigate T cells from progressing and rejected tumor sites and to determine if abnormal TCR {zeta} chain expression was related to lack of TIL effector function. Our studies show that TCR {zeta} expression is equivalent in both T cell populations regardless of effector function. These data support the work of Levi and Srivastava in which they reported that signaling abnormalities did not account for tumor-induced immune suppression (20).

Since TCR {zeta} chain expression was similar in both T cell populations, we speculated that differences in Vß expression might account for the disparate effector function of TIL and TRL. However, analysis of freshly isolated TIL and TRL revealed similar TCR Vß usage in both populations. In order to determine if the enhanced cytotoxicity seen after in vitro stimulation correlated with the expansion of TCR Vß1 and 8 bearing T cells, which predominated in vivo, TCR Vß usage was re-examined after MLTC. The data demonstrated that the percent of T cells bearing these TCR Vß decreased. Although the number of TCR Vß1 and 8 bearing T cells decreased after the MLTC, this decrease may have been the result of an increase in other TCR Vß bearing T cells. However, there did not appear to be a predominantly used TCR Vß in either the TIL or TRL population following in vitro stimulation. One explanation for this is that there may have been a loss of the TCR Vß1 and 8 bearing T cells that were not capable of responding to the tumor cells in vitro. If a population of TCR Vß1 and 8 bearing T cells died in culture, then the remaining cells could appear more oligoclonal upon examination of their CDR3 regions. An examination of the CDR3 regions did not reveal whether a population of tumor-reactive cells was growing in culture. There was an increase in clonality in the TCR Vß1 compartment. Yet, these clones were more frequent in the TIL population, which showed less cytolysis than the TRL population, making it difficult to determine their role in response to the tumor cells. Unfortunately, a TCR Vß1-specific antibody was not available at the time of the study for blocking experiments. CDR3 analysis within the TCR Vß8 compartment of the TIL and TRL showed a decrease in clonality. These data could indicate that either cytolysis after the MLTC is not restricted to one or two Vß families or the increased cytolytic activity following in vitro culture may be due to removal from an in vivo inhibitory environment. With regard to the first concept, the only way to identify the cells responsible for the cytolytic activity would be to deplete each subset until the cytolysis disappears. Since antibodies to all of the Vß TCRs were not yet available, this option was not feasible. In support of the second concept, we have noticed that an MLTC in the presence of transforming growth factor-ß, which inhibits maturation of precursor cytotoxic T lymphocytes to cytotoxic T lymphocytes, still resulted in increased cytolytic activity by the TIL population (data no shown). These data suggest that cytotoxic T lymphocytes were present in the TIL population in vivo and only showed tumor cytolysis after in vitro culture.

Although we have been able to block 15% of the TRL cytotoxicity against the EMT6 tumor cells using a TCR Vß8-specific antibody, we have also found that Vß8 TCR bearing T cell clones, grown in culture with repeated tumor cell stimulation, showed no cytolytic activity (data not shown). This suggests that the Vß8 TCR bearing T cells, which exhibit cytolytic activity, are not the cells that grow in vitro. Therefore, the only way to determine which T cells are responding to the tumor cells in vivo would be to deplete mice of individual T cell populations and determine which population, when depleted, inhibits the ability to reject tumors during concomitant tumor immunity. As for reasons stated above this is not currently feasible.

The current study has investigated several areas of interest in tumor immunology. First, these data suggest that a decrease in TCR {zeta} chain expression was not responsible for the inability of TIL to eradicate progressing tumors or their lack of cytolytic activity. Secondly, we have shown that the most predominant T cell clones present in progressing tumors were also the most predominant clones present in rejected tumor sites. Following secondary in vitro stimulation there was no expansion of T cells bearing these TCR, yet an increase in cytolytic function occurred. These data are reminiscent of Mackensen's study in which TCR Vß 16 predominated freshly analyzed TIL, yet a TCR Vß13 clone grew and responded to the autologous tumor in vitro (2). Further examination revealed the TCR Vß13 clone was present in the freshly analyzed TIL sample, yet was not a predominantly used TCR Vß. Collectively Mackensen's study and the data presented here suggest that the predominant TCR Vß used by lymphocytes infiltrating regressing or rejected tumors may not represent the tumor-reactive T cells which grow in culture or respond to the autologous tumor in vitro.


    Acknowledgments
 
The authors would like to thank Anita Choudhary for critically reviewing the manuscript, and helpful comments and discussions. Contract grant sponsors: the Arizona Disease Control Research Commission (9501 to E. T. A. and 5-038 to J. J. M.) and the National Science Foundation (MCB9406280 to J. J. M.).


    Abbreviations
 
MLTC mixed lymphocyte tumor culture
TIL tumor infiltrating lymphocyte
TRL tumor ejecting lymphocyte

    Notes
 
Transmitting editor: M. M. Davis

Received 26 October 1999, accepted 18 January 2000.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Ferradini, L., Mackenson, A., Genevee, C., Bosq, J., Duvillard, P., Avril, M. and Hercend, T. 1993. Analysis of T cell receptor variability in tumor-infiltrating lymphocytes from a human regressive melanoma. J. Clin. Invest. 91:1183.[ISI][Medline]
  2. Mackensen, A., Ferradini, L., Carcelain, G., Triebel, F., Faure, F., Viel, S. and Hercend, T. 1993. Evidence for in situ amplification of cytotoxic T-lymphocytes with antitumor activity in a human regressive melanoma. Cancer Res. 53:3569.[Abstract]
  3. Akporiaye, E. T., Kudalore, M., Stevenson, A. P., Kraemer, P. M. and Stewart, C. C. 1988. Isolation and reactivity of host effectors associated with the manifestation of concomitant tumor immunity. Cancer Res. 48:1153.[Abstract]
  4. Akporiaye, E. T. and Kudalore, M. K. 1989. Implantation of a gelatin-sponge as a model for effector recruitment. Cancer Immunol. Immunother. 29:199.[ISI][Medline]
  5. Kurt, R. A., Park, J. A., Panelli, M. C., Schluter, S. F., Marchalonis, J. J., Carolus, B. and Akporiaye, E. T. 1995. T lymphocytes infiltrating sites of tumor rejection and progression display identical Vß usage but different cytotoxic activities. J. Immunol. 154: 3969.[Abstract/Free Full Text]
  6. Mizoguchi, H., O'Shea, J. J., Longo, D. L., Loeffler, C. M., McVicar, D. W. and Ochoa, A. C. 1992. Alterations in signal transduction molecules in T lymphocytes from tumor-bearing mice. Science 258:1795.[ISI][Medline]
  7. Zea, A. H., Curti, B. D., Longo, D. L., Alvord, W. G., Strobl, S. L., Mizoguchi, H., Creekmore, S. P., O'Shea, J. J., Powers, G. C., Urba, W. J. and Ochoa, A. C. 1995. Alterations in T cell receptor and signal transduction molecules in melanoma patients. Clin. Cancer Res. 1:1327.[Abstract]
  8. Finke, J. H., Zea, A. H., Stanely, J., Longo, D. L., Mizoguchi, H., Tubbs, R. R., Wiltrout, R. H., O'Shea, J. J., Kudoh, S., Klein, E., Bukowski, R. M. and Ochoa, A. C. 1993. Loss of T-cell receptor {zeta} chain and p56lck in T-cells infiltrating human renal cell carcinoma. Cancer Res. 53:5613.[Abstract]
  9. Franco, J. L., Ghosh, P., Wiltrout, R. H., Carter, C. R. D., Zea, A. H., Momozaki, N., Ochoa, A. C., Longo, D. L., Sayers, T. J. and Komschlies, K. L. 1995. Partial Degradation of T-cell signal transduction molecules by contaminating granulocytes during protein extraction of splenic T cells from tumor-bearing mice. Cancer Res. 55:3840.[Abstract]
  10. Jorgensen, J. L., Esser, U., Groth, B., Reay, P. A. and Davis, M. M. 1992. Mapping T-cell receptor-peptide contact by variant peptide immunization of single-chain transgenics. Nature 355:224.[ISI][Medline]
  11. Sensi, M. and Parmiani, G. 1995. Analysis of TCR usage in human tumors: a new tool for assessing tumor-specific immune responses. Immunol. Today 16:588.[ISI][Medline]
  12. Malissen, M., Minard, K., Mjoisness, S., Kronenberg, M., Goverman, J., Hunkapillar, T., Prystosky, M. B., Yoshikai, Y., Fitch, F., Mak, T. W. and Hood, L. 1984. Mouse T cell antigen receptor: structure and organization of constant and joining gene segments encoding the ß polypeptide. Cell 37:1101.[ISI][Medline]
  13. Romero, P., Casanova, J., Cerottini, J., Maryanski, L. and Luescher, I. F. 1993. Differential T cell receptor photoaffinity labeling among H-2Kd restricted cytotoxic T lymphocyte clones specific for a photoreactive peptide derivative. labeling of the {alpha}-chain correlates with J{alpha} segment usage. J. Exp. Med. 177:1247.[Abstract]
  14. Osman, G. E., Toda, M., Kanagawa, O. and Hood, L. E. 1993. Characterization of the T cell receptor repertoire causing collagen arthritis in mice. J. Exp. Med. 177:387.[Abstract]
  15. Rosenberg, S. A. 1996. Development of cancer immunotherapies based on identification of the genes encoding cancer regression antigens. J. Natl. Cancer Inst. 88:1636.
  16. Belldegrun, A., Kasid, A., Uppenkamp, M., Topalian, S. L. and Rosenberg, S. A. 1989. Human tumor infiltrating lymphocytes: analysis of lymphokine mRNA expression and relevance to cancer immunotherapy. J. Immunol. 142:44520.
  17. Schwartzentruber, D. J., Solomon, D., Rosenberg, S. A. and Topalian, S. L. 1992. Characterization of lymphocytes infiltrating human breast cancer: specific immune reactivity detected by measuring cytokine secretion. J. Immunother. 12;1.[ISI][Medline]
  18. Peoples, G. E., Davey, M. P., Goedegebuere, P. S., Schoof, D. D. and Eberlein, T. J. 1993. T cell receptor Vß2 and Vß6 mediate tumor-specific cytotoxicity by tumor-infiltrating lymphocytes in ovarian cancer. J. Immunol. 151:5472.[Abstract/Free Full Text]
  19. Yoshizawa, H., Chang, A. E. and Shu, S. 1991. Specific adoptive immunotherapy mediated by tumor-draining lymph node cells sequentially activated with anti-CD3 and IL-2. J. Immunol. 147:729.[Abstract/Free Full Text]
  20. Levey, D. L. and Srivastava, P. K. 1995. T cells from late tumor-bearing mice express normal levels of p56lck, p59fyn, ZAP-70, and CD3 {zeta} despite suppressed cytolytic activity. J. Exp. Med. 182:1029.[Abstract]




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