In chronic lymphocytic leukemia (CLL), monoclonal B cells expand and accumulate in the blood, bone marrow, spleen and secondary lymphoid organs in response to incompletely defined cellular and molecular interactions involving multiple cell types within the tumor microenvironment (TME). Bidirectional interactions of CLL cells with components of the TME - mesenchymal stromal cells, myeloid cells, T cells, and matrix - are critical for leukemic cell survival and proliferation. Here, we focused on understanding how two non-leukemic cell types within the TME, T lymphocytes and myeloid-derived suppressor cells (MDSCs), interact to create a TME supportive of a CLL clone.
In CLL, it has been suggested alterations in CD4+ and CD8+ T cell ratios and functions are responsible for a non-inflammatory and immunosuppressive cytokine TME, with expansions of Th2s and Tregs. Conversely, higher numbers of Th17 and CD8+ cells are associated with longer survival. MDSCs are not well studied in CLL, although existing reports suggest that their expansion suppresses T cell proliferation in vitro. Here we studied the effects of MDSCs and CLL B cells on T cell numbers and function.
First, we identified a correlation between blood T cells and MDSCs in 56 CLL patients. Absolute numbers of MDSCs correlated with total numbers of CD3+, CD4+ and CD8+ cells (P= 0.002, < 0.001, < 0.001 and Spearman r = 0.44, 0.646, = 0.61, respectively). However, the correlation of MDSC subtypes with CD4+ and CD8+ cells differed: monocyte-like MDSCs (mMDSCs) associated with CD4+ cells (P < 0.001, Spearman r = 0.73) whereas granulocyte-like MDSCs (gMDSCs) associated with CD8+ cells (P= 0.008; Spearman r = 0.45).
Moreover, in a subset of 18 CLL patients tested, MDSC numbers directly correlated with the levels of Th2 (P= 0.049, Spearman r = 0.470), Th17 (P= 0.040, Spearman r = 0.488), Tc17 (P= 0.018, Spearman r = 0.550) and Tc22 (P= 0.010, Spearman r = 0.587) cells. When dividing MDSCs into subsets, gMDSCs exhibited a similar correlation except for the absence of Th2 and the presence of a correlation with Th22 (P= 0.033, Spearman r = 0.505). Conversely, mMDSCs positively associated with Th1 (P= 0.024, Spearman r = 0.549) and Tc1 (P= 0.019, Spearman r = 0.569) cells.
We next addressed the functional effect of MDSCs on naïve CD4+ cell differentiation using samples from 16 CLL patients and 10 healthy, age-matched controls (HCs). CD3+CD45RO-CD62L+ CD4+ lymphocytes were FACS-sorted and stimulated in vitro with anti-CD3/CD28 beads plus IL-2, and the percentages of CD4+ T cells producing various cytokines were quantified by intracellular flow cytometry. These results were compared with those obtained in the presence of mMDSCs (HLA-DRlo/CD11b+/CD33+/CD14+), gMDSCs (HLA-DRlo/CD11b+/CD33+/CD15+), monocytes (HLA-DRhi/CD11b+/CD14+) or CLL B lymphocytes (CD19+). On day 7, CLL T cells, cultured in the absence of other cells, generated a higher percentage of cytokine-producing cells than those from HCs (22% vs. 8%; P < 0.001). This difference was due to an increase in all cytokine-producing cells except classical Th17s producing IL-17A. When CLL T cells were co-cultured with gMDSCs, a significant expansion of Th2 (IL-4+) (P= 0.044) and a trend in growth of IL-17F+ Th17cells (P= 0.101) was found. In contrast, co-culture of CLL T cells with mMDSCs led to a reduction in Th22 (IL-22+) (P= 0.005) and Treg (FoxP3+) (P= 0.006) cells and to a trend towards expansion of Th2 (IL-4+) cells. Co-cultures of CLL T cells with monocytes, similarly to mMDSCs, led to an expansion of Th2 (IL-4+) cells (P= 0.01) and a reduction in Treg (FoxP3+) cells (P= 0.002). Finally, co-culturing naïve CD4+ lymphocytes with CLL B cells had a different and unexpected effect, promoting the expansion of Th1 (IFNγ+) cells (P= 0.033) and Th22 (IL-22+) T cells (P= 0.052).
In summary, the absolute number of blood T cells, CD4+ and CD8+ subsets, and MDSCs are linked, although associations of T cell subsets differ between mMDSCs and gMDSCs. MDSCs and their subsets influence naïve T cell differentiation differently, in general leading to expansion of Th2 (IL-4+) cells. In contrast, CLL B cells had a different effect on T cell polarization, preferentially expanding Th1 (IFNγ+) cells. Thus, it appears that myeloid cells and especially MDSCs are responsible for polarization of the T cell compartment to Th2 which is said to be dominant in CLL patients.
Kolitz: Gilead, Magellan, and Novartis: Honoraria; Gilead, Novartis, and Seattle Genetics: Other: Travel Support; Boehringer Ingelheim, Cantex, Erytech, and Millennium: Research Funding; Celgene, Jazz: Equity Ownership; Gilead, Magellan, Novartis, Pharmacyclics, and Seattle Genetics: Consultancy. Barrientos: Pharmacyclics LLC, an AbbVie Company: Consultancy, Research Funding; AbbVie: Consultancy, Research Funding; Janssen: Consultancy; Gilead: Consultancy, Research Funding. Stamatopoulos: Novartis SA: Research Funding; Abbvie: Honoraria, Research Funding; Gilead: Consultancy, Honoraria, Research Funding; Janssen Pharmaceuticals: Honoraria, Research Funding.
Asterisk with author names denotes non-ASH members.
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