Mechanism of T cell tolerance induced by myeloid-derived suppressor cells

Abstract

Ag-specific T cell tolerance plays a critical role in tumor escape. Recent studies implicated myeloid-derived suppressor cells (MDSCs) in the induction of CD8(+) T cell tolerance in tumor-bearing hosts. However, the mechanism of this phenomenon remained unclear. We have found that incubation of Ag-specific CD8(+) T cells, with peptide-loaded MDSCs, did not induce signaling downstream of TCR. However, it prevented subsequent signaling from peptide-loaded dendritic cells. Using double TCR transgenic CD8(+) T cells, we have demonstrated that MDSC induced tolerance to only the peptide, which was presented by MDSCs. T cell response to the peptide specific to the other TCR was not affected. Incubation of MDSCs with Ag-specific CD8(+) T cells caused nitration of the molecules on the surface of CD8(+) T cells, localized to the site of physical interaction between MDSC and T cells, which involves preferentially only TCR specific for the peptide presented by MDSCs. Postincubation with MDSCs, only nitrotyrosine-positive CD8(+) T cells demonstrated profound nonresponsiveness to the specific peptide, whereas nitrotyrosine-negative CD8(+) T cells responded normally to that stimulation. MDSCs caused dissociation between TCR and CD3zeta molecules, disrupting TCR complexes on T cells. Thus, these data describe a novel mechanism of Ag-specific CD8(+) T cell tolerance in cancer.

Figure 1. CD8⁺ T-cell tolerance caused by MDSC

A. Experimental protocol of MDSC-induced CD8⁺ T-cell tolerance. T cells isolated from OT-1 mice were transferred i.v. into naïve C57BL/6 mice. Two days later these mice were intravenously injected either with PBS (T cells alone group) or 3-4×10⁶ MDSC from EL-4 tumor-bearing (T cells + MDSC group) and at the same time were immunized s.c. with 100 μg of specific peptide SIINFEKL in IFA. Ten days later LN cells were collected or in some mice, re-immunized with specific peptide. The antigen specific CD8⁺ T cells from the LN were later evaluated. B. Mice were treated as described above. LN cells were collected and were restimulated in the presence of specific (S.P.) or control (C.P.) peptides. Number of IFN-γ producing cells was scored in ELISPOT assay. Results presented as Average±SD. Six experiments with the same results were performed. C. Typical example of staining with SIINFEKL pentamer. The experiment was performed as described in Fig. 1A. On day 3, after MDSC transfer and immunization with specific peptide, lymph node cells were collected and stained with anti-CD8 antibody conjugated with APC and SIINFEKL pentamer conjugated with PE. As a control of specific binding, CD4⁺ T cells were evaluated. D. LN cells isolated from mice after adoptive transfer of OT-1 T cells were labeled with anti-CD8 and anti- H-2Kb – SIINFEKL/Kb pentamer. Results presented as proportion of pentamer positive cells among CD8⁺ cells. Each time point includes 3 mice. E. Absolute number of CD8⁺ T cells and CD8⁺pentamer⁺ T cells was evaluated 3 days after immunization. Total number of cells per lymph node is shown. F. OT-1 T cells (5×10⁶) were labeled with 20μM CFSE and then adoptively transferred into naïve recipient mice. A day later these mice were injected with MDSC cells (5×10⁶) from EL-4 tumor-bearing mice and immunized s.c. with OT1 peptide. LNs were isolated 3 days later, stained with anti-CD8, and anti- H-2Kb - SIINFEKL pentamer and analyzed by flow cytometry.

Figure 2. MDSC don't induce TCR signaling in CD8⁺ T cells

A,B. B3Z T-cell hybridoma was cultured with mature bone marrow derived DCs or MDSCs loaded with OT1 peptide. A. Typical example of β-galactosidase activity measured in live (propidium iodide negative) cells by using fluorescein di-β-D-galactopyranoside (lower right quadrant). B. Cumulative results of three performed experiments. C,D. Calcium influx in T cell incubated with MDSC and DCs. C. OT-1 T cells were incubated with OT1 peptide loaded DCs or MDSCs and labeled with two calcium sensitive dyes fluo-4 or Fura-red. Ca²⁺ influx was measured as a ratio of fluorescence emitted by the two dyes – Fluo-4/Fura-red. D. Ca²⁺ influx after the addition of ionomycin. E. OT1 specific CD8⁺ T cells were cultured with peptide-loaded DCs or MDSC or both. Cells were stained with antibodies against CD8⁺ T cells, pCD3ζ, p56^lck, pZAP-70 and pERK1/2 and their expression measured in CD8⁺ T cells at different time points by flow cytometry. F. Peptide loaded DCs and MDSC were mixed together at 1:1 ratio and incubated with OT-1 T cells. The level of pZAP70 was evaluated either immediately (top panel) or after 3 hr of incubation.

Figure 3. Disruption of association between CD3ζ and TCR

OT-1 transgenic splenocytes were cultured with MDSC from EL-4 tumor-bearing C57BL/6 mice for 48 hr in the presence of the OT1 peptide (10μg/ml). 48hr later T-cells were isolated and analyzed. A. Cell surface FACS staining of T cells labeled with anti TCR-β anti-Vα2 mAbs or anti-Vβ5 mAb. B. Post-nuclear lysates of purified T cells from cultures containing either T cells alone or T cells with MDSC were subjected to IP-FCM. Native TCR/CD3 complexes were immunoprecipitated with mAbs specific for either CD3ζ (mAb H146) or Vβ5 (mAb MR9-4), and captured complexes were probed in parallel with PE-conjugated non-specific Hamster Ig, or mAbs specific for Thy1.2, CD3ζ (mAb 6B10), TCRβ (mAb H57), or Vα2 (mAb B20.1). Histogram fluorescence and the geometric MFIs displayed reflect the relative quantity of subunits in the complexes. Two experiments with the same results were performed. C. The relative CD3ζ content in the complexes was determined by dividing TCR β or α probe geometric MFI by that of ζ. Three experiments with the same results were performed. D. FRET assay. MDSC and OT-1 splenocytes were cultured for 48 hr with specific peptide and then stained with anti-CD8-PE or anti-CD3-PE antibodies (electron donor) and anti-TCRVα2-APC (electron acceptor). Cells were analyzed by confocal microscopy for FRET sensitized emission (FRET-SE) as described in Methods. FRET-SE was calculated in 10-16 regions with high intensity of fluorescence on different T cells and Mean ± SD are shown.

Figure 4. MDSC induces tolerance to only one specific TCR in T cell expressing dual TCR

A,B. Splenocytes were isolated from DT mice and cultured with MDSC isolated from EL-4 tumor-bearing mice at 1:4 ratio in the presence of 10μg/ml OT1 (A) or gp33 peptides (B). As control, splenocytes were cultured with specific peptide without MDSC. After 48 hr incubation T cells were purified and mixed at 1:5 ratio with splenocytes from naïve C57BL/6 mice and stimulated with either control, OT1 or gp33 peptides. The number of IFN-γ producing cells was evaluated in quadruplicates in an ELISPOT assay. Each experiment was performed three times. * - statistically significant (p<0.05) differences between control CD8⁺ T cells and T cells incubated with MDSC. C. T cells were isolated from DT mice and were transferred into naïve C57BL/6 recipients. MDSC isolated from tumor-bearing mice were injected two days later and mice were immunized once with OT1 peptide at day 2 and a second time 6 days later with 100 μg OT1 or gp33 peptide in IFA. Control mice received no MDSC and were immunized on day 2 and 6 days later with OT1 or gp33 peptide. Seven days after the second immunization response of LN cells to restimulation with control (CP), OT-1 or gp33 peptides was evaluated in an ELISPOT assay. Each group included three mice. * - statistically significant (p<0.05) differences between cells stimulated with control and specific peptides. D. DT cells were cultured with or without MDSC from EL-4 tumor-bearing in the presence of the 10μg/ml OT1 peptide. After 48hr splenocytes were washed, rested in serum-free medium for 90 min and then incubated with DCs loaded with OT1 or gp33 peptides. Cells were labeled with anti-CD8, anti-pZAP-70 and p56^lck and their expression evaluated using flow cytometry in triplicates. Mean ± SD are shown. * - statistically significant (p<0.05) differences between control T cells and T cells incubated with MDSC. E. Mice were implanted s.c. with 2×10⁵ EG7 or EL-4 tumor. Fifteen days later when tumor reach 1 cm in diameter mice were injected i.v. with 5×10⁶ DT T cells. Seven days after injection LN cells were isolated stimulated with control, OT1, or gp33 peptides and response was evaluated in ³[H]-thymidine assay. Mean ± SD are shown. * - statistically significant difference between cells stimulated with OT1 and gp33 peptides.

Figure 5. CD8⁺ T cell tolerance is specific for tumor-associated antigens

A. Antigen-specific CD8⁺ T cells after immunization with rNeu-derived peptide. BALB/c mice were primed with 100 μg of plasmid pEC_1–170^neu, followed immediately by electroporation of the injected area. Two weeks later, 100μg of synthetic peptide rNEUp66 was injected intravenously in combination with 50 μg of anti-CD40 mAb and 50 μg of Poly IC. Eight days after the last immunization, spleens were harvested and labeled with NEUp66-specific tetramer and anti-CD8 antibody and were evaluated by flow cytometry. CD8⁺ T cells were gated. B-C. Mice were injected with 2 × 10⁵ 66.3 and A2L2 cells. 10 days later mice were injected i.v. with 5×10⁶ rNEU specific T cells isolated from immunized mice. Seven days later the lymph node cells were restimulated with rNEUp66 peptide (SP) or control peptide (CP). IFN-γ production was evaluated in ELISPOT assay (B) and proliferation by ³[H]-thymidine uptake (C). LN cells were restimulated with specific (SP) or control (CP) peptides (B, C) or CD3/CD28 antibodies (D). T-cell proliferation was measured by ³[H]-thymidine uptake. Results presented as Mean±SD from four mice. * - statistically significant (p<0.05) differences between the groups.

Figure 6. MDSC causes nitration of the surface molecules on CD8⁺ T cells

A-E. DT T cells were isolated and labeled with OT-1 specific APC conjugated anti-Vα2 TCR antibody (magenta), gp33 specific Alexa 555-conjugated anti-Vβ 8.1 TCR antibody (red) and Alexa 488-conjugated anti-nitrotyrosine (NT) antibody (green). A. Typical staining of double transgenic CD8⁺ T cells is shown (left – gp33 TCR, right OT-1 TCR). B Double transgenic T cells were stimulated with DC loaded with OT-1 peptide. Arrow denotes polarization of Vα2 TCR. C. Double transgenic T cells were incubated with MDSC and OT-1 peptide. Arrows represent regions of positive NT staining and site of Vα2 TCR polarization. Bar = 5 μm. D, E. Double transgenic T cells were incubated for 48 hr with MDSC isolated from EG-7 tumor-bearing mice and then stained with APC conjugated anti-Vα2 TCR antibody (magenta) and Alexa 488-conjugated anti-nitrotyrosine (NT) antibody (green) (D) or PE-Cy5 conjugated anti-Vβ 8.1 TCR antibody (red) and Alexa 488-conjugated anti-nitrotyrosine (NT) antibody (green). Bar = 5 μm. E. Typical example of cell sorting gates of NT positive CD8⁺ T cells. MDSC from EL-4 tumor-bearing mice were cultured for 48 hr with splenocytes from OT-1 transgenic mice in the presence of the SIINFEKL (10μg/ml). 48 hr later, cells were stained with DAPI, anti-CD8 Alexa 647 and anti-nitrotyrosine Alexa 488 and sorted by flow cytometry. F. MDSC from EL-4 tumor-bearing mice were cultured for 48 hr with splenocytes from OT-1 transgenic mice in the presence of the OT1 peptide (10μg/ml). 48 hrs later cells were sorted and added at 1:5 ratio to naïve C57BL/6 splenocytes and cultured for 24, 48, or 72 hr prior to stimulation for 3 days with specific OT-1 peptide (SP) in a ³[H]-thymidine incorporation assay. 2ng/ml of IL-2 was added to the 48 and 72 hr cultures. Each experiment was performed in triplicate and repeated twice. Mean ± SD are shown. H. MDSC from EL-4 tumor-bearing mice were cultured for 48 hr with splenocytes from OT-1 transgenic mice in the presence of the SIINFEKL (10μg/ml). 48 hr later, cells were stained with DAPI, anti-CD8 Alexa 647, anti-Vα₂ APC and CD8⁺Vα₂⁺ cells were sorted. In control, cells were stained with DAPI, anti-CD8 and isotype control IgG and all CD8⁺ T cells were sorted. Cells were then cultured with naïve splenocytes at 1:10 ratio with 2ng/ml IL-2. These cells were rested for 24 h or 48h and then stimulated with control or specific (SIINFEKL) peptide and evaluated in triplicates in ³[H]-thymidine incorporation proliferation assay. J. Double transgenic T cells were incubated with MDSC for 48 hr in the presence of SIINFEKL peptide. CD8⁺ NT⁺ or NT^- cells were sorted as described above, mixed with naïve splenocytes and stimulated with either OT1 or gp33 peptides. The number of IFN-γ producing cells was evaluated in quadruplicates in ELISPOT assay.