Distinct Phenotypic and Functional Immunological Alterations Characterize the Peripheral Blood Compartment of Patients with Diffuse Large B Cell Lymphoma (DLBCL).

The impact of tumor presence on the immune competence of the host may be of particular relevance in the case of B cell neoplasia, as the tumor itself derives from immune system components; moreover, immune effector mechanisms are directly involved in determining the efficacy of chemioimmunoterapies.

It has been previously noted that alterations of the absolute number of circulating monocytes and lymphocytes (peripheral blood monuclear cells, PBMC) are a of prognostic relevance in diffuse large B cell lymphoma (DLBCL).

To analyze the phenotypic and functional asset of PBMC in DLBCL at diagnosis, and to evaluate possible correlations of the immunological profile with tumor biological traits and patient's clinical features.

We compared 30 consecutive newly diagnosed DLBCL patients with 21 healthy, age- and sex-matched controls for: 1) absolute number (/μL) and percentage (over PBMC) of monocytes, B cells, T cell (CD4+, CD8+, CD4+CD8+ double positive, CD4-CD8- double negative, CD56+ T cells, and FOXP3+CD25^bright regulatory T cells), and Natural Killer (NK) cell subsets (CD56^dim, CD56^bright, CD16+), measured by cell blood count and multi-parameter flow cytometric (FACS) analysis; 2) functional capability of individual T and NK cell subsets, by assessing the frequency of Interferon-gamma (IFN-γ) expressing cells and cytotoxic granule-containing cells; 3) natural and CD16-dependent NK cytotoxic activity, by ⁵¹Cr release assay, and 4) plasma concentration of selected cytokines, as evaluated with Bioplex.

DLBCL patients showed several quantitative and functional alterations of the PBMC compartment. DLBCL patients showed a higher absolute monocyte number (p=.001), and a lower lymphocyte count (p=.001), thus resulting in a strongly reduced lymphocyte/monocyte ratio (p<.0001). Lymphocyte reduction was mostly accounted for by a selective decrement of CD4+ T cells (p=.007) and B lymphocytes (p=.001), while the absolute number of CD8+ T cells and NK cells was not affected. As a result, the relative CD4/CD8 ratio decreased (p=.009), and NK percentage increased (p=.032). Neither the absolute number nor the percentage of CD4+CD25^brightFOXP3+ regulatory T cells were significantly different between DLBCL and controls. The phenotypically skewed profile of DLBCL patients was associated with functional alterations of selected peripheral blood lymphocyte subpopulations. The percentage of IFN-γ producing cells was increased in CD4+ (p=.005) and CD8+ (p=.005) T cell subsets (evaluated upon 6-hr PMA/ionomycin stimulation), but not in NK cell and innate-like (CD56+) T cell populations. The frequency of Granzyme B-expressing cells (as a measure of cytotoxic granule content) was markedly increased in CD4+ (p=.001) and CD8+ (p<.0001) T subsets, as well as in CD56^dim (p=.028) and CD56^bright(p<.0001) NK subsets. Noteworthy, natural and CD16-dependent NK cytotoxic activities were unchanged. The plasma levels of IL-6 and IL-10 were significantly higher in DLBCL (p<.0001 and p=.009, respectively). Some PBL alterations differently correlated with the DLBCL Germinal-Genter-B-type (GCB-type) vs non GCB-type and with the presence of bulky disease or extranodal involvement.

Our findings show that the phenotypic and functional profile of PBMC in DLBCL is deeply altered, with the lymphocyte compartment showing traits of chronic activation. We hypothesize that the functional deregulation may contribute, through a mechanism of altered turnover and/or trafficking, to the selective CD4+ T-cell lymphopenia in DLBCL patients. Noteworthy DLBCL histological subtypes and some clinical features correlated with defined immunological alterations. This suggests that peripheral blood lymphocytes might be valuable bio-sensor of lymphoma heterogeneity and of tumor-host crosstalk.

No relevant conflicts of interest to declare.