B lymphocytes can be activated to act as antigen presenting cells to promote anti-tumor responses

Abstract

Immune evasion by tumors includes several different mechanisms, including the inefficiency of antigen presenting cells (APCs) to trigger anti-tumor T cell responses. B lymphocytes may display a pro-tumoral role but can also be modulated to function as antigen presenting cells to T lymphocytes, capable of triggering anti-cancer immune responses. While dendritic cells, DCs, are the best APC population to activate naive T cells, DCs or their precursors, monocytes, are frequently modulated by tumors, displaying a tolerogenic phenotype in cancer patients. In patients with cervical cancer, we observed that monocyte derived DCs are tolerogenic, inhibiting allogeneic T cell activation compared to the same population obtained from patients with precursor lesions or cervicitis. In this work, we show that B lymphocytes from cervical cancer patients respond to treatment with sCD40L and IL-4 by increasing the CD80+CD86+ population, therefore potentially increasing their ability to activate T cells. To test if B lymphocytes could actually trigger anti-tumor T cell responses, we designed an experimental model where we harvested T and B lymphocytes, or dendritic cells, from tumor bearing donors, and after APC stimulation, transplanted them, together with T cells into RAG1-/- recipients, previously injected with tumor cells. We were able to show that anti-CD40 activated B lymphocytes could trigger secondary T cell responses, dependent on MHC-II expression. Moreover, we showed that dendritic cells were resistant to the anti-CD40 treatment and unable to stimulate anti-tumor responses. In summary, our results suggest that B lymphocytes may be used as a tool for immunotherapy against cancer.

Fig 1. B lymphocytes from cancer patients can be activated by treatment with sCD40L and IL-4.

Viable 90% enriched B lymphocytes from PBMCs from cervical cancer patients (cancer) or controls (control), were incubated with 10 μg/ml sCD40L and 50 ng/ml IL-4 for 24 hours. After harvesting, cells were labeled with anti-CD19, CD80, CD86 and HLA-DR and analyzed by flow cytometry, where 10⁵ events were acquired in a FACSCanto (BD Biosciences, Carlsbad, CA). For data analyzes, we excluded debris and doublets and gated the MHC-II, CD80⁺ and CD86⁺ populations on the CD19⁺ cells. We compared B lymphocytes from cancer patients (cancer) and controls (control), and within these two groups, sCD40L and IL-4 treated (+) and untreated cells (-). (A) Expression of MHC-II shown as median of fluorescence intensity (MFI); 100% of the cells were positive for MHC-II expression. (B) Percentage of the CD80⁺CD86⁺ population. Bars indicate groups with significant different results, based on ANOVA two-way test, where p<0.05 was elected as accepted. A total of 18 samples from patients with cancer and 8 samples from controls were used in this experiment.

Fig 2. Tumors induce tolerance in secondary lymphoid organs, but not through B lymphocytes.

(A) Diagram of the experimental design, where we harvested cells from C57Black/6 donors: naïve, with TC-1 tumors or immunized against tumor antigens; and transplanted into RAG1^-/- mice previously inoculated with TC-1 cells. After transplant, we measured tumor growth every other day for up to 21 days. (B) Tumor growth kinetics in RAG1^-/- mice transplanted with T lymphocytes isolated from lymph nodes from naïve, (Naïve T), HPV16 E6 and E7 peptides immunized (Immunizaed T), or TC-1 tumor bearing (Tumor T). nr—not reconstituted. One to 3 million lymphocytes were transplanted per mouse. Experimental groups of at least 6 mice (the experiments were repeated 3 times, so a total of at least 18 mice were tested); * indicates p<0.05, tested by Mann-Whitney U test. Experimental groups of at least 4 mice; * indicates p<0.05, tested by Mann-Whitney U test. We observed similar results when comparing the kinetics using the area under the curve as data for ANOVA testing.

Fig 3. Activation of B lymphocytes with anti-CD40.

(A) B lymphocytes sorted from tumor bearing C57Black/6 mice were treated ex vivo with 100 ng/ml anti-CD40 and analyzed by intracellular staining against the indicated phospho-proteins and flow cytometry; cells were stimulated for 30 min prior to harvesting. Results are the average of median fluorescence intensity of 3 independent experiments (indicated); (B) analysis of the expression of activation markers in peripheral lymph node cell suspensions determined by flow cytometry after 14 to 16 hours treatment. A representative experiment of 3 independent ones is shown (each experiment with at least 3 mice per group), and the average and standard deviation of the frequencies of the double-positive populations is shown in each dot-plot. Differences between control and treated cells were tested by t-test, * indicates p<0.05. (C) and (D) CellTrace labeled T lymphocytes from tumor bearing donors were incubated with sorted B lymphocytes from tumor bearing donors treated or not with 100 ng/ml anti-CD40 for 16 hours, in a 1:1 ratio. Phytohemagglutinin, (PHA) (1:100), or 5 μg/ml HPV16 E6 and E7 peptides (pep), were added to the cultures. Four days later, cells were harvested labeled with antibodies for CD4, CD8, CD154 and CD137 and analyzed by flow cytometry. The ratios of the percentage of CellTrace dim cells from cultures with anti-CD40 treated and control B lymphocytes (C) and expression ratio of CD154 or CD137, or both (DP) in cultures with treated and control B lymphocytes (D) are displayed. Ratios of three independent experiments, each one in biological triplicates (cells from 3 different mice, and our cultures were also plated in triplicates). The dashed lines indicate ratio value 1, where activation was the same in the two different conditions. The black solid short line, in C, indicates the proliferation ratio of T cells in neat cultures treated with PHA and compared to untreated T cells.

Fig 4. Tumor growth kinetics in mouse chimeras.

Lymphocytes isolated from C57Black/6 mice were transplanted into RAG1^-/- mice previously injected with 5x10⁴ TC-1 cells. One to 3 million lymphocytes were transplanted per mouse. When mixed, B or DC and T lymphocytes were transplanted in a 1:1 ratio. (A) T lymphocytes from tumor bearing donors, Tumor T, were injected alone or with B lymphocytes from tumor bearing donors activated with anti-CD40 or control: /CD40 tumor B or /control tumor B. (B) T cells from tumor bearing donors, Tumor T, were injected alone or with B cells from naïve donors, activated with anti-CD40 or control: /CD40 naive B or /control naive B. (C) T lymphocytes from naïve donors, Naïve T, were injected into the recipients, alone or with B lymphocytes from tumor bearing donors activated with anti-CD40 or control: /CD40 tumor B and /control tumor B. Experimental groups of 4 to 5 mice (the experiments were repeated 2 times, so a total of at least 8 to 10 mice were tested). Differences between groups was tested by Mann-Whitney U test, the tumor growth kinetics had experimental groups of at least 6 mice; * indicates p<0.05. Significant differences between groups had the same pattern when tested by ANOVA using as data the areas under the curves.

Fig 5. A. Chimeras with dendritic cells, DCs.

(A) DCs were isolated from tumor bearing C57Black/6 donors and stimulated or nor with anti-CD40 for 16 hours: /CD40 tumor DC and /control tumor DC, respectively. Cells were washed and mixed with T lymphocytes also from tumor bearing donors, Tumor T, injected into RAG1^-/- recipients. As controls, we had a group transplanted with anti-CD40 treated B (Tumor T/CD40 tumor B) lymphocytes and T lymphocytes from tumor bearing donors, a group transplanted with only T lymphocytes (Tumor T) and non reconstituted RAG1^-/- group, n.r. (B) Tumor volume in chimeras transplanted with T and B lymphocytes from tumor bearing donors (tumor T and tumor B) that were C57Black/6 (WT) or MHC-II^-/-. B lymphocytes were treated with anti-CD40 (CD40) or untreated cells (control) (Tumor T/CD40 tumor B and Tumor T/control tumor B). Tumor growth was monitored for 15 to 16 days. Each experimental group had 4 to 6 mice. Differences between groups were tested by ANOVA two way. The tumor growth kinetics had experimental groups of at least 6 mice; * indicates p<0.05.

Fig 6. Changes in tumor infiltrating T lymphocyte populations.

Flow cytometry analyzes of tumor cell suspensions from chimeras transplanted with T cells from tumor bearing donors alone (Tumor T) or together with anti-CD40 treated or control B lymphocytes from tumor bearing donors (/control Tumor B and /CD40 tumor B, respectively) or with anti-CD40 treated splenic dendritic cells (/CD40 tumor DC). Single cell suspensions were labeled with anti-CD45, CD11b, CD4, CD8, CD25, fixed, permeabilized and labeled with anti-Foxp3 and analyzed by flow cytometry. To identify the lymphocytes, after debris and doublets exclusion, we gated cells in the CD45⁺CD11b^- population, then CD4 or CD8 cells, and within the CD4⁺ population, selected CD25⁺Foxp3⁺ cells as regulatory T cells. Data represents the ratio between total CD4 or CD8 T cells and CD4 regulatory T cells. Tumor growth was monitored for 15 to 16 days. Each experimental group had 4 or 5 mice. Data was analyzed by ANOVA; * indicates p values <0.05.