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Journal of Leukocyte Biology logoLink to Journal of Leukocyte Biology
. 2011 Feb;89(2):291–300. doi: 10.1189/jlb.0610333

A caveat for T cell transfer studies: generation of cytotoxic anti-Thy1.2 antibodies in Thy1.1 congenic mice given Thy1.2+ tumors or T cells

Kyle C McKenna *,†,1, Rodolfo D Vicetti Miguel *,‡, Kelly M Beatty *, Richard A Bilonick *
PMCID: PMC3024899  PMID: 20959413

Thy 1.1 congenic mice given Thy 1.2+ tumors or T cells generate cytotoxic anti-Thy 1.2 antibody responses that delete transferred Thy 1.2+ T-cells.

Keywords: CD90, antibody, adoptive transfer

Abstract

Thy1.1 congenic B6.PL mice were used to simultaneously monitor Thy1.2+ E.G7-OVA tumors transplanted in the a.c. of the eye and i.v.-transferred tumor-specific Thy1.2+ CTLs to determine mechanisms that inhibit the tumoricidal activity of CTL responses in mice with established ocular tumors. Transferred CTLs were systemically deleted in mice with established ocular tumors. However, this deletion was not a unique mechanism of immune evasion by ocular tumors. Rather, development of Thy1.2+ tumors in the eye or skin of B6.PL mice generated cytotoxic anti-Thy1.2 antibodies that eliminated a subsequent Thy1.2+ T cell transfer. Anti-Thy1.2 immune responses in B6.PL mice were influenced by the route of antigen administration, as the serum concentration of cytotoxic anti-Thy1.2 antibodies was 92-fold greater in mice with eye tumors in comparison with mice with skin tumors. In addition, anti-Thy1.2 immune responses were detected in B6.PL mice given naïve Thy1.2+ T cells i.p. but not i.v. Anti-Thy1.2 responses were augmented in B6.PL mice with ocular Thy1.2+ EL-4 tumors that did not express OVA, suggesting immunodominance of OVA antigen over Thy1.2. Thy1.1+ T cells given i.p. was not immunogenic in Thy1.2 congenic mice. These data reaffirm that the introduction of antigens in the a.c. induces robust antibody responses. Experimentation using allotypic differences in Thy1 between donor cells and recipient mice must consider cytotoxic anti-Thy1 antibody generation in the interpretation of results.

Introduction

The method of adoptive transfer of T cells from TCR transgenic mice into recipient mice has made possible the characterization of antigen-specific T cell responses during infection [1], tolerance induction [2, 3], and malignancy [4]. Transferred T cells are easily identified by flow cytometric analysis using peptide/MHC tetramers [5] or by using mAb to discern allotypic differences between TCR transgenic mice and recipient mice in cell surface molecules such as Thy1/CD90 [6] and CD45 [7].

The Thy1.2 allotype is also expressed by E.G7-OVA tumors [4], the EL-4 thymoma is transduced to express chicken OVA as a surrogate tumor antigen [8], which is frequently used to test tumor immunotherapies in mice [9]. Using Thy1.2 to enumerate E.G7-OVA tumors in Thy1.1 congenic B6.PL mice, we have shown that these tumors are rejected by OVA-specific CD8+ T cell responses when limited numbers are transplanted in the skin but grow progressively when placed in the a.c. of the eye [10]. Ocular tumor growth primes for OVA-specific CD8+ T cell responses capable of eliminating a subsequent E.G7-OVA tumor challenge in the skin or opposite eye [10]. Therefore, the immune response is not ignorant of ocular tumors or incapable of functioning within a site of immune privilege. Rather, established ocular tumors appear to create a microenvironment, which is resistant to the tumoricidal activity of CD8+ CTLs.

To better understand mechanisms of immune evasion by ocular tumors, we used Thy1.1 congenic B6.PL mice that were transferred with activated Thy1.2+ OVA-specific OT-I CTLs before and after E.G7-OVA tumor challenge in the a.c. of the eye. Surprisingly, we observed that OT-I CTLs were systemically deleted in mice with established ocular tumors. However, this T cell deletion was not a novel mechanism of immune suppression by ocular tumors. Instead, Thy1.2+ E.G7-OVA tumor growth generated robust cytotoxic anti-Thy1.2 antibody responses in B6.PL mice, which eliminated the subsequent Thy1.2+ T cell transfer. Cytotoxic anti-Thy1.2 antibodies were also generated when naïve Thy1.2+ T cells were injected i.p. but not i.v. into Thy1.1 congenic mice. These data highlight a significant caveat for adoptive transfer studies exploiting allotypic differences in Thy1 molecules to monitor transferred T cells.

MATERIALS AND METHODS

Experimental animals

Male and female C57Bl/6PL (B6.PL, H-2b, Thy1.1/CD90.1+), C57Bl/6 (B6, H-2b, Thy1.2/CD90.2+), C57Bl/6J-TgN (TCR-1) (OT-I, H-2b, Thy1.2/CD90.2+), B6.129S2-Igh-6tm1Cgn/J (μMT, H-2b, Thy1.2/CD90.2+), B6.129P2-B2mtm1Unc/J (β2M−/−, H-2b, Thy1.2/CD90.2+), and Balb/CJ mice were purchased from The Jackson Laboratory (Bar Harbor, ME, USA). 2C mice (2C, H-2b, Thy1.2/CD90.2+) [11], B6.PL, OT-I, and μMT were bred and maintained in the animal facilities at the University of Pittsburgh (PA, USA). μMT mice were backcrossed with B6.PL mice to generate Thy1.1 congenic B6.PL μMT mice. All procedures on animals were approved by the Institutional Animal Care and Use Committee at the University of Pittsburgh in adherence to the provisions of the Association for Research in Vision and Ophthalmology Statement for the use of Animals in Ophthalmic and Vision Research.

Tumor cell lines

EL-4 (H-2b, Thy1.2/CD90.2+) and EL-4 transduced to express OVA (E.G7-OVA) were purchased from American Type Culture Collection (Manassas, VA, USA) and were grown in SGM (RPMI 1640 supplemented with 10% FBS, 2 mM L-glutamine, 1 mM sodium pyruvate, 50 μM 2-ME, gentamycin, penicillin, and streptomycin) at 37°C in a 5% CO2 atmosphere. All cell lines were maintained free of mycoplasma, and E.G7-OVA were cultured in 1.0 mg/ml G418 sulfate to maintain the expression of the transfected OVA gene.

Tumor cell injections

a.c. injections (104) and i.d. skin injections (106) were performed as described previously [10].

CTL generation and adoptive transfer

Naive splenocytes from OT-I mice (4×106) were stimulated with 0.1 μg/ml OVA peptide 257–264 (SIINFEKL) in individual wells of a 24-well plate in a total volume of 2 ml SGM. Following incubation for 3 days at 37°C in a 5% CO2 atmosphere, Ficoll separation was performed to remove dead cells, and the number of live cells was determined by trypan blue exclusion. No further enrichment of the purified, activated effector splenocytes was performed, as >95% of live cells after Ficoll separation were Thy1.2+ CD8+ OVA257–264 Kb tetramer+ T cells (data not shown). OVA-specific lytic activity by OT-I-stimulated splenocytes (OT-I CTL) was measured in a 51Cr release assay as described previously [10], and OT-I CTL specifically lysed only targets expressing OVA (data not shown). 2C CTLs were generated by stimulating naïve 2C splenocytes (2.0×106) with irradiated Balb/C splenocytes (4.0×106) in 2 ml SGM in individual wells of a 24-well plate for 3 days. For adoptive transfer experiments, OT-I or 2C CTLs (3×106 cells) were injected i.v. via the tail vein in 200 μl PBS and were tracked in recipient B6 mice using Thy1.2 as a congenic marker by flow cytometric analysis.

Tissue processing and flow cytometry

Mice were killed by asphyxiation with CO2, and then, LNs and/or spleens were removed and rendered into a single cell suspension by pressing the tissues through a nylon mesh screen (70 μm). RBCs were lysed with Tris-buffered ammonium chloride and washed with HBSS, and then, cells were resuspended in FACS buffer (PBS+1% FBS) or PBS at cell concentrations determined by cell counts in a Vi-CELL XR counter (Beckman-Coulter, Miami, FL, USA).

Tumor-bearing eyes were rendered into a single cell suspension in 0.4 ml FACS buffer or SGM as described previously [10], and eye suspensions (0.1 ml) or leukocytes (splenocytes, peripheral blood leukocytes, or LN cells) were added to individual wells of a 96-well plate for staining. FcRs were blocked with purified anti-CD16/32 mAb (BD PharMingen, San Diego, CA, USA). Eye suspensions were stained with anti-CD45, anti-CD8α, anti-CD11b, and anti-Thy1.2 antibodies (BD PharMingen). Splenocytes or LN cells (106) were stained with allophycocyanin-conjugated anti-Thy1.2 antibodies. In some experiments, a fixable violet dead cell stain (Invitrogen, Carlsbad, CA, USA) was added during incubation with primary antibodies. Following incubation, wells were washed and fixed with Cytofix/Cytoperm reagent (BD PharMingen). A FACSAria flow cytometer (Becton Dickinson, San Jose, CA, USA) was used to collect events for flow cytometric analysis, which was performed using FACSDiva (BD PharMingen) and FlowJo (Tree Star, Ashland, OR, USA) software. Ocular tumor cell numbers were quantified as described previously [10] with some modifications. Specifically, Count Bright absolute counting beads (Invitrogen) were used to collect half of each collagenase-digested eye sample from the known, initial sample volume.

In vivo killing assay

LN cells and splenocytes from naïve B6 or B6.PL mice were labeled with 2.5 μM Cell Tracker Green™ CFDA-succinimidyl ester (Invitrogen), according to the manufacturer's instructions, and then 2.0 × 107-labeled cells were i.v.-injected into Thy-1 disparate recipient mice via the tail vein. In some experiments, mice were given i.p. injections of anti-CD4 (0.2 mg) and/or anti-CD8 (0.2 mg) antibodies to deplete CD4 or CD8 T cells prior to CFSE transfer. Rat IgG (0.4 mg) was administered to some mice as a control for antibody administration. One to 4 days after transfer of CFSE-labeled target cells, nontumor draining LNs, spleens, or blood were removed, processed, and then stained with anti-Thy1.2 antibodies for flow cytometric analysis. From gated CFSE+ cells, the number of Thy1.2+ and Thy1.2-negative cells was determined. The following formula was then used to determine the percent specific lysis of Thy1.2+ cells: 100 × [(number of Thy1.2-negative targets×A)–(number of Thy1.2+ targets)/(number of Thy1.2-negative targets×A)]. A = (number of Thy1.2+/number of Thy1.2-negative targets) in control, untreated mice.

Lytic antibody detection

Blood was isolated by cardiac puncture, incubated at 37°C for 30 min to promote clotting, and centrifuged to isolate serum, and then, serum was incubated at 56°C for 30 min to inactivate complement. Splenic T cells from B6 or B6.PL mice, isolated by T cell enrichment columns (R&D Systems, Minneapolis, MN, USA), were used as Thy1.2+ and Thy1.2-negative targets, respectively. Targets were labeled with 51Cr for 1 h, washed with HBSS, and then resuspended in FACS buffer. Serum or control anti-Thy1.2 antibody (BD PharMingen) was then added to targets (5.0×103) contained within an individual well of a 96-well plate and incubated for 30 min at 8°C. Wells were then washed with FACS buffer and cytotoxicity media (Cedarlane, Hornby, Ontario, Canada) before the addition of complement contained in cytotoxicity medium (Cedarlane). Following 30 min incubation at 37°C, plates were centrifuged, and then, 51Cr release was measured in culture supernatants using a TriLux β plate reader (Perkin Elmer/Wallac, Turku, Finland). Triton-X was added to targets to determine maximal lysis, and the amount of 51Cr release in targets incubated with complement alone was used as a measure of spontaneous lysis. Percent lysis of target cells was determined using the following formula: 100 × [(experimental 51Cr release–spontaneous release)/(maximal–spontaneous release)]. Eight threefold dilutions of serum beginning at 1:10 were performed to determine the dilution at which 50% of targets were lysed (LD50).

Statistical analysis

Groups were compared using GraphPad (La Jolla, CA, USA) Prism v4 software by ANOVA, t tests, or Mann-Witney U-tests, depending on the normality of data, and P < 0.05 was considered statistically significant. The Pearson product-moment correlation coefficient was used to determine the linear association between the square root of the number of OT-I T cells within the eye and spleen of the same mouse. Using the R statistical programming language, a sigmoid nonlinear mixed effects model [12, 13] with random intercepts was fitted to the relationship between the logarithm of serum dilution and the logit of the proportion of lysis of target cells to determine LD50 values.

RESULTS

Transferred CTL are deleted in B6.PL mice with E.G7-OVA tumors

To better understand the mechanisms that inhibit the tumoricidal activity of CTLs in established ocular tumors, Thy1.1 congenic B6.PL mice were used to simultaneously monitor Thy1.2+ CD8-negative E.G7-OVA tumors and i.v.-transferred Thy1.2+ CD8+ OVA-specific CTLs by flow cytometric analysis (Fig. 1A). As shown in Fig. 1B, a marked reduction in ocular tumor burden was observed in mice transferred with in vitro-generated OT-I CTL on the same day as tumor challenge in comparison with nontransferred control mice. Tumor elimination was associated with OT-I CTL infiltration of ocular tumors. In contrast, tumor burden was not significantly reduced when CTLs were transferred 7 days after a.c. tumor challenge, although OT-I CTLs were observed within some ocular tumors (Fig. 1C). In both experiments, tumor burden and CTL infiltration of ocular tumors were measured 4 days after CTL transfer.

Figure 1. Deletion of transferred Thy1.2+ CTLs in Thy1.1 congenic mice with Thy1.2+ tumors.

Figure 1.

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(A) Expression of Thy1.2, Thy1.1, and CD8 by tumors, T cells, and utilized mouse strains. Thy1.1 congenic B6.PL mice received an i.v. transfer of Thy1.2+ CD8+ OT-I CTL before (B) or after (C) E.G7-OVA tumor challenge in the a.c. of the eye. Plots display expression of Thy1.2 and CD8α by CD45+ cells in collagenase-digested eyes isolated 4 days after CTL transfer. (D) The number of transferred OT-I CTLs was determined in the spleens of B6.PL mice with ocular and skin tumors. Each symbol represents a measurement from an individual mouse, pooled from two to three independent experiments, and horizontal bars indicate the mean. One-way ANOVA with Dunnett's post-test comparison between the mean number of splenic OT-I CTLs (3.1±1.6×106) in mice transferred on the same day as tumor challenge and in mice with established eye tumors or established skin tumors indicated differences between means of 1.9 × 106 (95% CI for the mean difference of 0.62–3.26×106 for Day 7 ocular tumors and 0.50–3.4×106 for Days 10–11 skin tumors); **P < 0.01. (E) Mice with established Day 7 ocular E.G7-OVA tumors were injected with OT-I T cells. Four days later, the number of OT-I T cells in the eye and spleen was determined. Regression lines with eye OT-I T cell numbers (dashed line) or spleen OT-I T cell numbers (solid lines) as dependent variables are shown.

Surprisingly, we observed that OT-I CTL numbers in the spleens of mice transferred 7 days after tumor challenge in the eye were reduced significantly (mean reduction=1.6-fold) in comparison with mice that received a CTL transfer on the same day as tumor challenge (Fig. 1D). A similar twofold reduction in splenic CTL numbers was observed when CTLs were transferred into mice with established skin tumors (Fig. 1D). As OT-I CTL transfer promotes skin tumor regression in C57Bl/6 mice [14], splenic CTL depletion could have been a result of CTL recruitment to the site of tumor development. However, a direct correlation between the number of splenic CTLs and the number of CTL-infiltrating ocular tumors was observed (Fig. 1E), which indicated that reduced numbers of splenic CTLs could not be explained by increased CTL accumulation within ocular tumors. Decreased numbers of transferred CTLs were also observed in blood, liver, lung, and LNs of ocular tumor-bearing mice (data not shown), suggesting that transferred CTLs were systemically deleted or deleted upon encountering a high dose of OVA expressed within established tumors.

OVA expression by ocular tumors is not required for deletion of OT-I CTLs

To determine whether OVA expression by tumors was required for deletion of transferred OT-I CTLs, B6.PL mice were injected in the a.c. or i.d. in the skin with the parental tumor cell line EL-4, which does not express OVA, and then, mice received an OT-I CTL transfer 7 or 10 days later (Fig. 2A and D). Four days after CTL transfer, the mean percentages of OT-I CTLs in blood (Fig. 2B) and the mean number of splenic OT-I CTLs (Fig. 2C) in mice with ocular tumors were significantly reduced 41-fold and 26-fold, respectively, in comparison with transferred control mice without ocular tumors. Transferred CTL deletion was also observed in mice with established skin tumors (Fig. 2E and F) but at a much lower magnitude (twofold CTL depletion within the blood and in the spleen). These data indicated that OVA, expressed directly by tumors or cross-presented as processed peptides on APCs, was not required for CTL deletion. Therefore, growth of Thy1.2+ tumors in Thy1.1 congenic mice may have induced anti-Thy1.2 immune responses, which eliminated the subsequent Thy1.2+ OT-I CTL transfer.

Figure 2. Tumor expression of OVA is not required for deletion of transferred OT-I CTLs.

Figure 2.

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B6.PL mice received an i.v. transfer of OT-I CTLs 7 days after EL-4 tumor challenge in the eye (A–C) or 10 days after tumor challenge in the skin (D–F). The percentage of OT-I T cells in the blood (B and E) and the number of OT-I T cells in the spleen (C and F) were determined 4 days later. Each symbol represents a measurement from an individual mouse. (B, C, and F) Horizontal bars indicate the median and (E) mean. (B, C, and F) The median percentage or median number of OT-I T cells in tumor-challenged mice was compared with unchallenged mice by a Mann-Whitney U-test. (C) The mean percentage of OT-I T cells in tumor-challenged mice was compared with unchallenged mice by a t test. (B and C) Data presented are from one representative experiment of three independent experiments with similar results. (E and F) Data presented are pooled from two independent experiments. *P < 0.05; **P < 0.01; ns, P > 0.05.

Thy1.2+ tumors transplanted in the a.c. induce adaptive anti-Thy1.2 immune responses

Thy1.1 and Thy1.2 are distinguished by single amino acid differences in arginine or glutamine, respectively, at Position 89 [15]. Therefore, the exquisite specificity of an anti-Thy1.2 immune response in Thy1.1 congenic mice would suggest recognition by a highly selective antigen receptor, for example, Ig or TCR, expressed by cells of the adaptive immune response. Clonal expansion of a limited number of Thy1.2-specific T or B cells should require time. Therefore, we monitored Thy1.2+ CTL numbers when CTLs were transferred 3, 5, or 7 days after ocular EL-4 tumor challenge (Fig. 3). Thy1.2+ 2C CTLs, which recognize H-2Kd, were used in this experiment to further confirm that OVA specificity was not required for deletion and that deletion was not unique to OT-I CTLs. The percentage of 2C CTLs in the blood (Fig. 3A) and the number of 2C CTLs in the spleen (Fig. 3B) of mice transferred 7 days after tumor challenge were significantly reduced ten- and 15-fold, respectively, in comparison with CTL-transferred mice without ocular tumors, which reproduced our previous observations. 2C CTLs were not deleted, however, in mice that were transferred after only 3 days of ocular tumor growth and were only slightly reduced after 5 days of tumor growth, which is consistent with the generation of an adaptive anti-Thy1.2 immune response that requires time to develop.

Figure 3. Influence of tumor growth on deletion of transferred OT-I T cells.

Figure 3.

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B6.PL mice received an i.v. transfer of Thy1.2+ 2C CTLs 3, 5, or 7 days after a.c. challenge with EL-4 tumors. Plots display the percentage of 2C cells within the blood (A) or the number of 2C cells within the spleen (B). Each symbol represents a measurement from an individual mouse, and horizontal bars indicate the mean. One-way ANOVA with Dunnett's post-test comparison between the mean percentage of 2C CTLs in blood (3.1±0.54) or mean number of splenic 2C CTLs (2.0±1.1×105) in tumor-free CTL-transferred mice and in mice with Day 7 ocular tumors indicated mean differences of 2.78 (95% confidence intervals for the mean difference of 1.6–4.0) for the percentage of OT-I CTLs in blood and 1.9 × 105 (95% confidence interval for the mean difference of 0.56–3.17×105) in the number of splenic OT-I CTLs. *P < 0.05; **P < 0.01. All other comparisons were not statistically significant.

To confirm further that anti-Thy1.2 immune responses mediated elimination of transferred Thy1.2+ CTLs in ocular tumor-bearing B6.PL mice, three different experimental designs were used. First, Thy1.2+ CTLs were transferred into tumor-bearing Thy1.2 congenic B6 mice, which express Thy1.2 as a self-antigen and therefore, are tolerant to Thy1.2. As shown in Fig. 4A, the number of transferred OT-I CTLs in spleens of B6 mice with and without ocular EL-4 tumors was equivalent, whereas B6.PL mice with ocular EL-4 tumors deleted transferred CTLs (mean reduction=139-fold). These data indicated that transferred CTLs were only deleted when Thy1.2 was expressed as a semiallogeneic minor histocompatibility antigen. In the next experiment, B6.PL mice, with and without ocular EL-4 tumors, received a transfer with Thy1.1+ OT-I CTLs (Fig. 4B), which were not deleted in these mice, indicating that the immune response induced by ocular EL-4 tumor growth was specific for Thy1.2 and not other antigens with shared expression by tumors and T cells. In a final set of experiments, an in vivo killing assay [16, 17] was performed using a uniformly CFSE-labeled population of naïve splenocytes/LN cells from Thy1.2 congenic B6 mice, which contained Thy1.2+ and Thy1.2-negative targets (Fig. 4C). B6.PL mice with ocular (Fig. 4C and D) or skin tumors (Fig. 4D) selectively deleted Thy1.2+ targets but not Thy1.2-negative targets, confirming that EL-4 tumor growth in B6.PL mice induced anti-Thy1.2 immune responses. The deletion of transferred Thy1.2+ targets was significantly greater in mice with ocular tumors than in mice with skin tumors (Fig. 4D), reproducing our original observations (Fig. 2).

Figure 4. Generation of anti-Thy1.2 immune responses in tumor-bearing Thy1.1 congenic mice.

Figure 4.

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(A) B6 mice and B6.PL mice were untreated or injected with EL-4 tumors in the a.c. of the eye. On Day 7, OT-I CTLs were injected i.v., and the mean number of OT-I CTLs in the spleen was determined by staining with OVA257–264 Kb tetramers or anti-Thy1.2 antibodies, respectively, 4 days later. Each symbol represents a measurement from an individual mouse, and horizontal bars indicate the mean. ANOVA with Tukey's comparison test indicated no statistically significant difference between splenic OT-I CTL numbers in tumor-free B6 and B6 mice with ocular tumors. The comparison of mean splenic OT-I CTL numbers in tumor-free B6.PL mice (4.96±2.72×105) and B6.PL mice with ocular EL-4 tumors indicated a statistically significant difference in means of 4.92 × 105 (95% CI for the mean difference of 1.32–8.52×105). (B) Untreated B6.PL mice and B6.PL mice with Day 7 ocular EL-4 tumors were transferred with Thy1.1+ OT-I (PL) CTL. Four days later, the number of transferred CTLs in the spleen was quantified. (C) Untreated and ocular EL-4 tumor-bearing B6.PL mice were i.v.-injected with CFSE-labeled splenocytes (SPL)/LN cells from B6 mice 7 days after tumor challenge. One day later, deletion of Thy1.2+ target cells was determined by flow cytometric analysis. Plots display the percentage of Thy1.2+ and Thy1.2-negative targets in gated CFSE+ cells from a representative sample. (D) An unpaired t test with Welch's correction was used to compare the mean in vivo deletion of Thy1.2+ target cells in mice with Day 7 tumors in the a.c. (93.6±2.7%) or Day 10 i.d. skin tumors (59.6±30.2%). Target cell deletion was measured 1–3 days after i.v. transfer of CFSE-labeled targets. The difference between means was 34.0 (95% CI for the mean difference of 10.75–57.37). Each symbol represents measurements from individual mice pooled from two independent experiments for each tumor treatment, and horizontal bars indicate the mean ** P < 0.01; ns, not statistically significant (P > 0.05).

Thy1.2 expression and in vivo growth characteristics of E.G7-OVA and EL-4 tumors

Thy1.1 congenic B6.PL mice with ocular EL-4 tumors demonstrated greater deletion of transferred Thy1.2+ target cells than B6.PL mice with ocular E.G7-OVA tumors (compare Figs. 1D with 2C and 4A). Therefore, we evaluated Thy1.2 expression by these tumor cell lines and their in vivo growth characteristics to determine whether EL-4 tumors displayed greater expression of Thy1.2 or grew at a faster rate in vivo, thereby increasing the expression of Thy1.2 protein via increased tumor cell numbers. As shown in Fig. 5A, EL-4 and E.G7-OVA demonstrated equivalent expression of Thy1.2 in vitro. In two independent experiments, ocular tumor numbers were comparable 7 days after tumor challenge in the a.c. (Fig. 5B and D), indicating similar in vivo tumor growth characteristics between E.G7-OVA and EL-4. Modest differences in the in vivo expression of Thy1.2 between E.G7-OVA and EL-4 tumors were observed but were inconsistent, as one experiment showed a 2.4-fold decrease in Thy1.2 expression by EL-4 tumors (Fig. 5C), whereas another experiment showed a 1.5-fold increase (Fig. 5E). These data indicated that differences in Thy1.2 expression or in vivo tumor growth could not explain the differences observed in anti-Thy1.2 responses between mice with ocular E.G7-OVA and EL-4 tumors.

Figure 5. Thy1.2 expression and in vivo growth characteristics of E.G7-OVA and EL-4 tumors.

Figure 5.

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(A) Thy1.2 expression by cultured tumor cell lines. Data are displayed as Thy1.2 mean fluorescence intensity (MFI), and each symbol indicates a separate experiment. Thy1.2 MFI was normalized to background fluorescence by tumor cells stained with an isotype control antibody. (B–E) In two independent experiments, B6.PL and Thy1.1 congenic μMT mice (B6.PL μMT) were challenged with 104 E.G7-OVA or EL-4 tumor cells in the a.c. of the eye. Seven days later, eyes were removed, collagenase digested, and then, tumors were enumerated (B and D), and expression of Thy1.2 (C and E) was determined immediately ex vivo by flow cytometric analysis. Each symbol represents measurements from an individual mouse. *P < 0.05; **P< 0.01; ns, not statistically significant (P > 0.05).

Role of T cells and B cells in anti-Thy1.2 immune responses in ocular tumor-bearing mice

To determine whether CD4+ or CD8+ T cells mediated anti-Thy1.2 immune responses in ocular tumor-bearing mice, these cell populations were eliminated with depleting antibodies prior to i.v. transfer of Thy1.2+ and Thy1.2-negative targets in the in vivo killing assay. Despite depletion of CD4 and/or CD8 T cells, Thy1.2+ targets were still eliminated in ocular tumor-bearing B6.PL mice, and this elimination was equivalent to what was observed in ocular tumor-bearing mice that did not receive depleting antibodies or were given nonspecific rat IgG (Fig. 6). These data indicated that anti-Thy1.2 immune responses were not mediated by conventional CD8+ or CD4+ T cells or CD4+ NK T cells.

Figure 6. B cells are required for the generation of anti-Thy1.2 immune responses in Thy1.1 congenic mice.

Figure 6.

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Thy1.1. congenic B6.PL or B cell-deficient μMT mice (B6.PL μMT) were challenged with EL-4 tumors in the a.c. of the eye. Certain mice were given indicated antibody treatments before CFSE-labeled Thy1.2+ and Thy1.2-negative target cells were transferred 7 days post-tumor challenge. The plot displays percent Thy1.2+ target deletion. Each symbol represents a measurement from an individual mouse pooled from three independent experiments, and horizontal bars indicate the mean. One-way ANOVA and Dunnet's post-comparison of the mean percent Thy1.2+ target deletion between control B6.PL mice with ocular EL-4 tumors (83.5±11.5) and μMT mice with EL-4 tumors indicated a significant difference in means of 81.13 (95% confidence interval for mean difference of 72.1–90.2). There was no statistically significant difference between the means of control mice and ocular tumor-bearing mice depleted of CD4 and/or CD8 T cells by antibodies. Control mice with ocular tumors were untreated or given rat IgG prior to transfer of Thy1.2+ and Thy1.2-negative targets; **P < 0.01; ns, not statistically significant (P > 0.05).

To determine whether B cells contributed to anti-Thy1.2 immune responses, B cell-deficient μMT mice, backcrossed onto a Thy1.1 congenic background, were challenged in the a.c. with EL-4 tumors or untreated, and the in vivo killing assay was then performed 7 days later. Thy1.2+ targets were not deleted in B cell-deficient mice with ocular tumors (Fig. 6), although growth of EL-4 tumors was equivalent to that observed in B6.PL mice (Fig. 5D). These data indicated that B cells played a critical role in the expression or generation of anti-Thy1.2 immune responses in tumor-bearing Thy1.1+ mice.

Humoral responses in B6.PL mice with Thy1.2+ tumors

To determine whether cytotoxic anti-Thy1.2 antibody responses were generated in EL-4 tumor-bearing B6.PL mice, serum was isolated from mice with ocular or skin tumors and tested for complement-dependent lysis of Thy1.2 target cells. As shown in Fig. 7A, Thy1.2+ but not Thy1.1+ targets were lysed by serum from B6.PL mice with ocular tumors when complement was added. Lysis of Thy1.2+ targets was negligible when serum from nontumor-bearing control B6.PL mice (Fig. 7A) or μMT mice with ocular tumors (data not shown) was used, confirming that tumor growth induced complement-fixing cytotoxic anti-Thy1.2 antibody responses. A sigmoidal nonlinear-mixed effects model with random intercepts was fitted to serum dilution curves from groups of mice with ocular (n=6) and skin tumors (n=6) to determine the serum dilution, where 50% of Thy1.2+ target cells were lysed (LD50; Supplemental Fig. 1). The median-predicted LD50 values for mice with ocular tumors were 92-fold greater than in mice with skin tumors (Fig. 7B), indicating that the magnitude of the anti-Thy1.2 antibody responses was greater in serum from mice with eye tumors, which is consistent with our data from the in vivo killing assay (Fig. 4D).

Figure 7. Cytotoxic anti-Thy1.2 antibodies in Thy1.1 congenic mice bearing Thy1.2+ tumors.

Figure 7.

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(A) Serum from B6.PL mice that were untreated or challenged with EL-4 tumors in the a.c. of the eye was monitored for the presence of cytotoxic anti-Thy1.2 antibodies. The plot shows lysis of indicated targets after incubation with serum at multiple dilutions and addition of complement. (B) LD50 values for B6.PL mice challenged with EL-4 tumors in the eye or skin. Each symbol represents a measurement from an individual mouse, and horizontal bars indicate the mean. An unpaired t test with Welch's correction was used to compare LD50 differences between mice with EL-4 tumors in the a.c. or i.d. in the skin, which indicated a 92-fold difference between the means (95% CI for the ratio of the means of 6.0–1424). **P < 0.01.

Anti-Thy1.2 antibody responses are not MHC class I-restricted

The generation of anti-Thy1.2 antibody responses in Thy1 disparate mice has been shown to require MHC compatibility between immunizing donor cells and recipient mice [18, 19]. These data could suggest antigenic competition between incompatible MHC and Thy1.2 or MHC restriction of antibody responses. MHC class I restriction was postulated, as anti-Thy1.2 antibody responses required compatibility at H-2 K and D regions [19]. To test directly whether anti-Thy1.2 antibody responses in our system were MHC-restricted, an in vivo killing assay was performed in tumor-bearing mice using Thy1.2+ and Thy1.2-negative splenocytes/LN cells from β2M−/− mice that lack classical and nonclassical MHC class I expression. As shown in Fig. 8, ocular tumor-bearing B6.PL mice deleted class I-deficient Thy1.2+ target cells from B2M−/− mice. These data indicated that anti-Thy1.2 antibody responses were not MHC class I-restricted.

Figure 8. Anti-Thy1.2 antibody responses are not MHC class I-restricted.

Figure 8.

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(A) Thy1.1 congenic B6.PL mice were challenged with EL-4 tumors in the a.c. Seven days later, mice received uniformly labeled CFSE+ Thy1.2+ and Thy1.2-negative target cells from β2M−/− mice. Percent-specific Thy1.2+ target deletion was determined 1–3 days later. Each symbol represents a measurement from an individual mouse pooled from two independent experiments. Deletion of Thy1.2+ MHC class I-expressing targets (from Fig. 4D) is shown for comparison ns, not statistically significant (P > 0.05).

Generation of anti-Thy1 immune responses to transferred splenocytes

The method of adoptive transfer of T cells from TCR transgenic mice into recipient mice that express allotypic differences in the Thy1 molecule is used extensively to track antigen-specific T cells in vivo. To determine whether adoptive transfer of Thy1.2+ T cells into B6.PL recipients could induce anti-Thy1.2 immune responses, we injected B6 splenocytes/LN cells into B6.PL mice via i.v., i.p., or i.d. routes, and then, an in vivo killing assay was performed with Thy1.2+ and Thy1.2-negative targets (Fig. 9). i.p. injection of B6 splenocytes generated anti-Thy1.2 immune responses that were equivalent in magnitude (60% specific lysis) to those generated by EL-4 tumor growth in the skin (compare with Fig. 4D). In contrast, B6 splenocyte injection via i.d. or i.v. routes consistently generated weak or undetectable anti-Thy1.2 responses. i.p. injection of Thy1.1 splenocytes from B6.PL mice into B6 mice also generated weak anti-Thy1.1 responses. These data indicate that Thy1.2 is immunogenic in B6.PL mice, whether expressed by naïve T cells or tumors, and that Thy1.1 expressed by splenocytes/LN cells is much less immunogenic in B6 mice.

Figure 9. Generation of cytotoxic anti-Thy1.2 immune responses after immunization with naïve Thy1.2+ T cells.

Figure 9.

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Thy1.1 congenic B6.PL (PL) or Thy1.2 congenic B6 mice were immunized with naïve Thy1 disparate T cells from B6 or B6.PL mice, respectively, by injection via the indicated routes. Seven days later, recipient mice received an injection with CFSE-labeled splenocytes from Thy1 disparate mice. Plots display percent-specific deletion of Thy1.2 or Thy1.1 targets. Each symbol represents a measurement from an individual mouse combined from two independent experiments for each group. One-way ANOVA with Tukey's comparison between percent-specific target deletion in B6.PL mice immunized via i.p. B6 splenocyte injection (59±40) indicated significant differences with the means of mice immunized by i.d. injection (−50.38; 95% confidence interval of −92.14 to −8.613) or i.v. injection (−47.35; 95% confidence interval of −89.11 to −5.588). A significant difference in means was also observed in comparison with B6 mice that were immunized i.p. with B6.PL splenocytes (−51.25; 95% confidence interval of −14.95 to −87.55) *P < 0.05, **P < 0.01; ns, not statistically significant (P > 0.05).

DISCUSSION

We demonstrate that growth of Thy1.2+ EL-4/E.G7-OVA tumors in the a.c. of the eye of Thy1.1 congenic B6.PL mice induces cytotoxic anti-Thy1.2+ antibody responses. As a result, transferred Thy1.2+ CTLs were eliminated rapidly in B6.PL mice with established EL-4 tumors. Therefore, experimentation aimed at determining the fate of transferred OVA-specific OT-I CTLs in mice with established ocular E.G7-OVA tumors should use Thy1.2+ B6 mice and monitor OT-I CTLs with OVA257–264 H-2Kb tetramers. Alternatively, OT-I mice can be backcrossed onto a Thy1.1 background for use in Thy1.1 congenic B6.PL mice. This strategy is ideal, as Thy1.1+ OT-I CTLs can still be monitored by tetramers, and Thy1.2 is retained as an exclusive marker of EL-4/E.G7-OVA tumors. Our data also indicate that Thy1.1 was less immunogenic than Thy1.2 in Thy1 disparate mice (Fig. 9). Therefore, experimental designs involving the transfer of T cells into Thy1 disparate recipients should use Thy1.1+ T cells.

At first glance, our data appear inconsistent with a previous report by Shrikant and Mescher [4], who did not observe deletion of transferred OT-I T cells in Thy1.1 congenic mice with E.G7-OVA tumors. Specifically, B6.PL mice received an i.v. transfer of OT-I T cells or were left untreated, and then, mice were injected with E.G7-OVA tumors 1 day later in the peritoneal cavity. The authors observed that OT-I T cells dramatically expanded in the peritoneal cavity 4 days after tumor challenge and then declined rapidly by Day 8. An additional transfer of OT-I T cells on Day 8 again resulted in expansion, not deletion, of the fresh OT-I T cells in these tumor-bearing mice, suggesting that tumor growth did not induce anti-Thy1.2 immune responses. However, more careful interrogation of these data reveals that the expansion of OT-I T cells, transferred into mice with Day 8 E.G7-OVA tumors, was twofold lower when mice did not receive a previous i.v. OT-I transfer. These data could suggest that i.v. administration of Thy1.2+ T cells tolerized the anti-Thy1.2 immune response in Thy1.1 congenic mice. Another explanation is that tumor growth in the peritoneal cavity does not induce anti-Thy1.2 immune responses. We did not test this hypothesis directly. However, we did observe that injection of naïve Thy1.2+ T cells in the peritoneal cavity of B6.PL mice induced anti-Thy1.2 immune responses (Fig. 9) comparable in magnitude with what we observed in mice with established skin tumors (Fig. 4). Therefore, Thy1.2 expression within the peritoneal cavity appears to be immunogenic. A third possibility is that the expression of OVA by the tumor may influence the generation of anti-Thy1.2 immune responses, as we demonstrated only a 1.6-fold deletion in transferred Thy1.2+ T cells in mice with established Day 7 E.G7-OVA tumors in the eye (Fig. 1), whereas transferred Thy1.2+ T cells were reduced ten- to 139-fold in mice with Day 7 parental EL-4 tumors that did not express OVA (Figs. 2–4). This profound difference in anti-Thy1.2 immune responses in tumor-bearing mice was not a result of differences in Thy1.2 expression or tumor growth characteristics of EL-4 and E.G7-OVA (Fig. 5), which suggests that OVA may be immunodominant to Thy1.2.

The magnitude of anti-Thy1.2 immune responses was also influenced by the site of tumor growth, as the concentration of complement-fixing anti-Thy1.2 antibodies in serum (Fig. 7) and corresponding deletion of transferred Thy1.2+ cells in vivo (Fig. 4) were greater in Thy1.1 congenic mice with ocular EL-4 tumors than in mice with EL-4 skin tumors. These data are consistent with a previous report that demonstrated that complement-fixing antibody responses to antigens expressed by P815 mastocytomas were greater in magnitude when P815 tumors developed within the eye in comparison with P815 tumor development in the skin [20]. It is important to note that EL-4 and P815 tumors grow progressively in the a.c. of the eye [10, 21], despite induction of these complement-fixing antibodies that are tumor-specific. Similarly, tumor-specific antibody responses are detected in patients with progressively growing uveal melanomas [22]. Therefore, the inability of these antibodies to influence ocular tumor growth may be explained by the expression of complement-regulatory proteins, including decay accelerating factor, membrane cofactor protein (CD46), CD55, and CD59, within ocular fluids [23] and by uveal melanomas [24].

We are not the first to describe the generation of cytotoxic anti-Thy1 antibody responses in Thy1 disparate mice. Indeed, 30 years ago, Lake and Douglas [18] and Gorzynski and Zaleski [19] independently demonstrated complement-dependent cytotoxic anti-Thy1.2 antibody responses in several different Thy1.1 congenic mouse strains immunized by i.v. injection with Thy1.2+ thymocytes. These data would indicate that transferred T cells should ultimately be deleted by complement-mediated lysis in Thy1 disparate recipient mice. However, many adoptive transfer experiments have effectively used allotypic differences in Thy1 to monitor i.v.-transferred T cells in vivo. Most often, Thy1.1+ T cells, isolated from spleens and LNs, are transferred into Thy1.2 congenic mice, and these transferred T cells persist long-term [6]. Similarly, i.v.-transferred Thy1.2+ T cells have been detected 90 days post-transfer in Thy1.1 congenic mice [25]. Therefore, thymocyte-expressed Thy1.2 may be more immunogenic than Thy1.2 expressed by splenocytes and LN cells.

The generation of anti-Thy1.2 antibody responses in Thy1 disparate mice was shown to require H-2 compatibility between donor Thy1.2+ thymocytes and recipient mice [18, 19] and varied in magnitude between Thy1.1 congenic strains of different haplotypes (H-2q>H-2f>H-2a>H-2b>H-2k) [19]. In addition, the two antigenic components (MHC molecules and Thy1.2) had to be expressed by the same cell [26]. These data were interpreted in two different ways: Gorzynski and Zaleski [19] favored the interpretation that anti-Thy1.2 antibodies were MHC class I-restricted. In contrast, Clark et al. [26] concluded that antigenic competition between Thy1.2 and immunodominant-incompatible MHC inhibited the generation of anti-Thy1.2 responses, as anti-Thy1.2 immune responses were abrogated when F1 donor thymocytes bearing compatible and incompatible MHC were used. Our data showing that anti-Thy1.2 antibody responses eliminated class I-deficient target cells (Fig. 8) provide strong evidence that these antibody responses are not MHC class I-restricted. Moreover, our observation that OVA expression by tumors decreased the anti-Thy1.2 immune responses supports the interpretation of antigenic competition.

What remains puzzling is that abrogation of anti-Thy1.2 antibody responses as a result of antigenic competition with histoincompatible MHC only occurred if both antigens were expressed on the same cell [18, 26]. The observation that anti-Thy1.2 antibody responses to cell-bound Thy1.2 were CD4-dependent suggested a hapten carrier effect [27]. Therefore, one possibility is that immunogenic microparticles [28] are actively released by thymocytes, which express both antigens. Irradiated thymocytes do not induce anti-Thy1.2 antibody responses in Thy1 disparate mice [26], which supports that antigen presentation by Thy1 disparate thymocytes is an active process. Therefore, B cells may simultaneously internalize Thy1.2 and incompatible MHC for processing and presentation via MHC class II to CD4+ Th cells. Differences in TCR affinity or antigen-specific T cell frequency could then favor MHC-specific Th responses, limiting T cell-mediated help for anti-Thy1.2 B cell responses. Antigenic competition could also occur at the level of Ig recognition of antigens expressed on microparticles. Similar to T cell responses, differences in Ig affinity or antigen-specific B cell frequency could favor the generation of an antibody response to one antigen and limit the response to the other antigen.

Differential release of microparticles by thymocytes, tumors, or splenocytes/LN cells may also explain the differences in magnitude of anti-Thy1.2 antibody responses when cell-bound Thy1.2 is administered via different routes and by different cell populations. Along those lines, D'Orazio and coworkers [29] have demonstrated that OVA-pulsed macrophages release OVA to prime B cells located across a transwell, only when macrophages are pretreated with TGF-β, which produces macrophages analogous to ocular macrophages. This phenomenon may have been mediated by microparticle release, but further experimentation is necessary to determine if microparticles are involved and whether microparticle release is greater in the eye than in other sites. Interestingly, microparticles are increased in patients with Sjogren's syndrome, an inflammatory condition of the eye that is antibody-mediated [30].

In conclusion, Thy1 molecules expressed by transferred T cells or tumor cells can be immunogenic in Thy1 disparate recipients under certain circumstances. Therefore, any experimentation using allotypic differences in Thy1 between donor cells and recipient mice must consider cytotoxic anti-Thy1 antibody generation in the design and interpretation of results.

ACKNOWLEDGMENTS

This work was supported by National Eye Institute (NEI) grants R01 EY018355 and P30-EY08098, The Eye and Ear Foundation of Pittsburgh, and an unrestricted grant from Research to Prevent Blindness Inc. The authors thank Nancy Zurowski for excellent assistance in flow cytometry and Leah Kaercher for her technical contribution.

The online version of this paper, found at www.jleukbio.org, includes supplemental information.

a.c.
anterior chamber
CI
confidence intereval
H0
null hypothesis
SGM
standard growth medium

AUTHORSHIP

K.C.M. designed most experiments, performed some experiments, interpreted results of all experimentation, performed some statistical analysis, wrote the manuscript, and was responsible for regulatory compliance. R.D.V.M. designed some experiments, performed some experiments, and interpreted results of some experimentation. K.M.B. performed most experimentation and interpreted some results. R.A.B. performed statistical analysis.

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