Expression of major histocompatibility complex (MHC) and costimulatory molecules in vitro was not different between 4T1.2 and 4T1.2-HER2 tumor cell lines
4T1.2-HER2 tumor cells expressed high levels of HER2 whereas 4T1.2 tumor cells were HER2-negative (Additional File 2: Figure S1). Both 4T1.2 and 4T1.2-HER2 cells were positive for MHC I molecules (H-2Kd and H-2Dd) and negative for MHC II (I-Ad) and costimulatory molecules (CD80 and CD86) (Additional File 2: Figure S1).
4T1.2 and 4T1.2-HER2 tumor cells had similar proliferation rates in vitro but different tumor growth patterns in vivo
4T1.2 and 4T1.2-HER2 tumor cells had similar in vitro proliferation rates (Fig. 1A). When inoculated orthotopically into wildtype BALB/c mice, 4T1.2 tumor (5x104 cells inoculated) demonstrated a rapid, continuous progression and mice became moribund at as early as day (D) 30 post tumor inoculation (Fig. 1B). Despite a 40-fold higher dose of tumor cell inoculation, 4T1.2-HER2 (2x106 cells inoculated) tumor development did not track the rapid progression as observed in the 4T1.2 model but consisted of three phases: (i) initial tumor growth, (ii) spontaneous tumor regression, and (iii) a second phase of tumor outgrowth or tumor rejection (Fig. 1C). In a separate experiment where mice were inoculated with different doses of 4T1.2-HER2 tumor cells, tumor recurrence rate during phase (iii) showed an increasing trend with increasing doses of tumor cell inoculation (χ2 test for trend, p = 0.076). Additionally, when inoculated into immunodeficient BALB/c scid mice, 4T1.2-HER2 tumor (5x105 cells inoculated) demonstrated a rapid, continuous progression and no spontaneous tumor regression was observed (Fig. 1D).
4T1.2-HER2 tumor growth was associated with changes in tumor-infiltrating effector and immunosuppressive cells
Female BALB/c mice were orthotopically inoculated with 2x106 4T1.2-HER2 cells and sacrificed at D6, 9, 12, 15 and 18 post tumor inoculation (n = 8/time point). Initial tumor growth peaked at D9 and was followed by spontaneous tumor regression (Fig. 2A). There was a significant difference in terminal tumor weight (Fig. 2B) (one-way ANOVA, F(4, 22) = 3.167, p = 0.034) and splenic cell number (Fig. 2C) (one-way ANOVA, F(4, 35) = 2.692, p = 0.047) across all time points. The number of total tumor-infiltrating immune cells (Fig. 2D) and infiltrating cells per gram of tumor (Fig. 2E) was not significantly different across all time points.
Among tumor-infiltrating effector cells, the percentage of tumor-infiltrating CD4+ (Fig. 3A) (Kruskal-Wallis test, KW = 14.35, p = 0.006) and CD8+ (Fig. 3B) (one-way ANOVA, F(4, 22) = 3.35, p = 0.028) T cells was reduced from D6 to D9 during the initial tumor growth period and increased after D9 as tumor regression occurred. The percentage of tumor-infiltrating CD49b+ NK cells (Fig. 3C) (one-way ANOVA, F(4, 20) = 55.01, p < 0.001) was reduced at D9-18 compared to D6. In the immunosuppressive compartment, the percentage of tumor-infiltrating Gr-1+/CD11b+ MDSCs (Fig. 3D) (one-way ANOVA, F(4, 22) = 4.962, p = 0.005) was reduced at D18, CD11b+/Ly6Clo/Ly6G+ gMDSCs (Fig. 3E) was unchanged, and CD11b+/Ly6Chi/Ly6G− mMDSCs (Fig. 3F) (Kruskal-Wallis test, KW = 13.79, p = 0.008) was reduced over time. Tumor-infiltrating CD4+:MDSC (Fig. 3G) (one-way ANOVA, F(4, 22) = 5.168, p = 0.004) and CD8+:MDSC (Fig. 3H) (one-way ANOVA, F(4, 22) = 5.481, p = 0.003) ratios were increased at D18.
Splenic CD4+ and CD8+ T cells demonstrated the same trend with tumor-infiltrating T cells and was reduced from D6 to D9 and increased after D9 (Additional File 2: Figure S2). In contrast to T cells, splenic MDSCs were increased from D6 to D9 and reduced after D9. As a result, splenic CD4+:MDSC and CD8+:MDSC ratios were reduced from D6 to D9 during the initial tumor growth period and increased after D9 as tumor regression occurred (Additional File 1: Figure S2). The distribution of other myeloid cells and Tregs in the spleen and the tumor are shown in Additional File 2: Figure S3 and Additional File 2: Figure S4, respectively.
4T1.2-HER2 tumor growth was associated with changes in tumor-infiltrating memory T cells
Within tumor-infiltrating T cells, the percentage of CD44+/CD62L+ central memory (TCM) cells within CD4+ (one-way ANOVA, F = 9.310, p < 0.001) and CD8+ (one-way ANOVA, F = 4.510, p = 0.008) T cells was increased over time. The percentage of CD44+/CD62L− effector memory (TEM) cells within CD4+ T cells was reduced at D15 compared to D6-9 (one-way ANOVA, F = 5.127, p = 0.005). The percentage of CD69+, CD25+, CD44−/CD62L+ naïve and CD44−/CD62L− effector cells within tumor-infiltrating CD4+ or CD8+ T cells was not significantly different between time points (Table 1).
Table 1
Temporal changes in tumor-infiltrating activated and/or memory T cells during 4T1.2-HER2 tumor growth.
Tumor | D6 | D9 | D12 | D15 | D18 | p-value |
% of CD4+ T cells | CD69+ | 7.12 ± 0.78 | 8.66 ± 0.43 | 8.82 ± 0.41 | 7.74 ± 0.80 | 8.25 ± 0.69 | 0.301 |
CD25+ | 9.79 ± 0.46 | 10.9 ± 0.54 | 9.83 ± 0.25 | 10.2 ± 0.59 | 11.5 ± 0.13 | 0.117 |
CD44−/CD62L+ (naïve) | 85.9 ± 2.38 | 83.1 ± 1.59 | 85.0 ± 1.50 | 85.5 ± 2.36 | 86.9 ± 0.41 | 0.494 |
CD44−/CD62L− (effector) | 7.20 ± 1.74 | 6.55 ± 0.93 | 6.01 ± 1.13 | 4.47 ± 1.45 | 2.92 ± 0.61 | 0.084 |
CD44+/CD62L+ (TCM) | 3.17 ± 0.12a | 6.77 ± 0.52b | 6.29 ± 0.28b | 8.37 ± 1.07b | 7.82 ± 0.68b | < 0.001 |
CD44+/CD62L− (TEM) | 3.73 ± 0.62a | 3.60 ± 0.34a | 2.70 ± 0.31a,b | 1.68 ± 0.25b | 2.39 ± 0.21a,b | 0.005 |
% of CD8+ T cells | CD69+ | 7.59 ± 0.80 | 8.20 ± 0.20 | 9.04 ± 0.29 | 7.46 ± 0.83 | 7.09 ± 0.27 | 0.156 |
CD25+ | 1.20 ± 0.28 | 1.97 ± 0.17 | 1.72 ± 0.18 | 1.96 ± 0.39 | 1.74 ± 0.05 | 0.203 |
CD44−/CD62L+ (naïve) | 89.3 ± 1.54 | 83.9 ± 1.27 | 84.3 ± 1.39 | 83.4 ± 2.90 | 86.6 ± 0.32 | 0.203 |
CD44−/CD62L− (effector) | 5.83 ± 1.02 | 7.61 ± 0.91 | 6.72 ± 1.12 | 6.56 ± 2.00 | 4.13 ± 1.15 | 0.294 |
CD44+/CD62L+ (TCM) | 3.62 ± 0.29a | 5.89 ± 0.71a,b | 6.01 ± 0.45a,b | 6.91 ± 0.69b | 7.04 ± 0.85b | 0.008 |
CD44+/CD62L− (TEM) | 1.42 ± 0.30 | 2.70 ± 0.24 | 2.99 ± 0.37 | 3.39 ± 0.84 | 2.47 ± 0.31 | 0.119 |
One-way ANOVA or Kruskal-Wallis test. Labeled means at each time point without a shared letter are significantly different. n = 3–7/time point. |
Consistent with tumor-infiltrating TCM cells, the percentage of splenic CD44+/CD62L+ TCM cells within CD4+ and CD8+ T cells was also increased over time. The distribution of other splenic effector and memory cell populations is shown in Additional File 1: Table S3.
4T1.2-HER2 tumor growth was associated with changes in tumor-infiltrating immune cells expressing effector molecules
To better understand the functional status of effector cells, we assessed the expression of effector molecules (IFNγ, IL-2, TNFα and perforin) within CD4+, CD8+ and CD49b+ cells, respectively. Among tumor-infiltrating effector cells, there was a significant difference in the percentage of IFNγ + cells within CD4+ T cells (Fig. 4A) (one-way ANOVA, F(4, 20) = 2.961, p = 0.045) across all time points. The percentage of IFNγ + cells within CD8+ T cells (Fig. 4B) was unchanged over time, and the percentage of IFNγ + cells within CD49b+ NK cells (Fig. 4C) (one-way ANOVA, F(4, 20) = 17.83, p < 0.001) was increased from D9 to D12 and reduced from D15 to D18. The percentage of IL-2+ cells within CD4+ T cells (Fig. 4D) (one-way ANOVA, F(4, 20) = 37.97, p < 0.001) was increased from D6 to D12 and reduced from D12 to D18, the percentage of IL-2+ cells within CD8+ T cells (Fig. 4E) (one-way ANOVA, F(4, 20) = 43.71, p < 0.001) was increased from D9 to D12 and reduced from D15 to D18, and the percentage of IL-2+ cells within CD49b+ NK cells (Fig. 4F) (Kruskal-Wallis test, KW = 15.65, p = 0.004) was reduced at D18. The percentage of TNFα+ cells within CD4+ T cells (Fig. 4G) was not significantly changed over time, the percentage of TNFα+ cells within CD8+ T cells (Fig. 4H) (one-way ANOVA, F(4, 20) = 4.350, p = 0.011) was reduced at D18 compared to D9, and the percentage of TNFα+ cells within CD49b+ NK cells (Fig. 4I) (one-way ANOVA, F(4, 20) = 7.442, p < 0.001) was reduced at D15-18 compared to D6. The percentage of perforin+ cells within CD8+ T cells (Fig. 4J) (one-way ANOVA, F(4, 20) = 6.278, p = 0.002) was increased at D15 compared to D6, followed by a reduction at D18. The percentage of perforin+ cells within CD49b+ NK cells (Fig. 4K) (one-way ANOVA, F(4, 20) = 5.852, p = 0.003) was reduced at D18 compared to D9-12.
Among splenic effector cells, the percentage of IFNγ + cells and IL-2+ cells within CD4+ T cells, CD8+ T cells and CD49b+ NK cells, respectively, was increased from D6 to D15, followed by a reduction at D18 (Additional File 2: Figure S5). The percentage of TNFα+ cells within CD4+ and CD8+ T cells was increased from D6 to D9, and the percentage of perforin+ cells within CD8+ T cells and CD49b+ NK cells was increased at D12-15 compared to D6 (Additional File 2: Figure S5).
4T1.2-HER2 tumor regression occurred concurrently with HER2-specific IFNγ production
To further characterize antigen-specific T cell response during the initial tumor growth and regression, we assessed IFNγ secretion by tumor-infiltrating (Table 2) and splenic (Additional File 1: Table S4) immune cells following ex vivo stimulation with an H-2Kd-restricted HER2 peptide, a control HA peptide, anti-CD3/anti-CD28 antibodies, or without stimulus, respectively. Differences between HER2 and HA peptide-stimulated groups at each time point were further assessed using paired t test or Wilcoxon test (a two-way ANOVA was not applicable due to non-normal distribution of the data) (Fig. 5 and Additional File 2: Figure S6). IFNγ secretion by tumor-infiltrating immune cells in HER2 group was higher than HA group at D12 (paired t test, p = 0.030) and D15 (Wilcoxon test, p = 0.063) (Fig. 5). IFNγ secretion by splenic immune cells in HER2 group was higher than HA group at D12 (Wilcoxon test, p = 0.063) (Additional File 2: Figure S6).
Table 2
Temporal changes in IFNγ secretion by tumor-infiltrating immune cells during 4T1.2-HER2 tumor growth.
Tumor | HER2 | HA | αCD3 + αCD28 | Media |
D6 | 108.7 ± 71.78 | 96.84 ± 69.76 | 199800 ± 38625 | 75.78 ± 35.91 |
D9 | 60.84 ± 41.52 | 12.80 ± 8.564 | 196966 ± 73138 | 7.309 ± 7.309 |
D12 | 315.7 ± 148.3 | 13.61 ± 7.986 | 326294 ± 88817 | 29.09 ± 12.32 |
D15 | 319.7 ± 222.7 | 150.5 ± 139.3 | 135152 ± 50084 | 153.5 ± 136.7 |
D18 | 102.6 ± 90.46 | 45.64 ± 40.28 | 40855 ± 34650 | 40.79 ± 31.37 |
Data are expressed in pg/mL. n = 3–6/time point. |
Tumor volume during the second phase of 4T1.2-HER2 tumor growth was correlated with the density of tumor-infiltrating immune cells and the percentage of effector and immunosuppressive cells
In mice that demonstrated the second phase of 4T1.2-HER2 tumor growth (recurrence), tumor volume was not correlated with the number of total tumor-infiltrating immune cells (Fig. 6A) but was negatively correlated with the number of tumor-infiltrating immune cells per cm3 of tumor (Fig. 6B) (r = 0.662, p = 0.052). In addition, tumor volume was not correlated with the percentage of tumor-infiltrating T cells (Additional File 2: Figure S7), but was positively correlated with the percentage of tumor-infiltrating CD11b+/Gr-1+ MDSCs (Fig. 6C) (r = 0.842, p = 0.004) and CD11b+/Ly6Clo/Ly6G+ gMDSCs (Fig. 6D) (r = 0.657, p = 0.055), and negatively correlated with the percentage of tumor-infiltrating CD11b+/Ly6Chi/Ly6G− mMDSCs (Fig. 6E) (r=-0.903, p < 0.001), CD4+:MDSC (Fig. 6F) (r=-0.774, p = 0.014) and CD8+:MDSC (Fig. 6G) (r=-0.776, p = 0.014) ratios.
Additionally, during the second phase of tumor growth or tumor rejection, tumor volume was positively correlated with splenic cell number and the percentage of splenic MDSCs, and negatively correlated with the percentage of splenic effector cells (Additional File 2: Figure S8). Further, splenic effector to immunosuppressive cell ratios were significantly higher in mice that rejected the tumor compared to mice that demonstrated the second phase of tumor growth (Additional File 2: Figure S9).