Phenotypically and Functionally Altered T Cell Compartment in DLBCL Patients at Diagnosis and Its Long-Term Modification upon Chemoimmunotherapy Regimen
Authors: Cox Maria Christina , Battella Simone , La Scaleia Raffaella , di Napoli Arianna , Pelliccia Sabrina , Porzia Alessandra , Mainiero Fabrizio , Monarca Bruno , Ruco Luigi , Santoni Angela , Palmieri Gabriella 
Published: October 2019

Abstract
Background: Peripheral blood lymphopenia at diagnosis and after treatment has been widely reported as a negative prognostic factor in DLBCL. This phenomenon, which reflects host systemic immunity, is still poorly characterized. Very recently, we have described a profound phenotypic and functional alteration of the peripheral NK cell compartment in newly-diagnosed DLBCL patients at diagnosis and upon R-CHOP chemoimmunotherapy, and suggested that the complex dynamics of the immune system plays a pivotal role in the response to chemoimmunotherapy. Since the role of T cell-dependent memory and effector capabilities in long-term cure of chemoimmunotherapy-treated cancer patients is increasingly appreciated, we have focused our studies on their modifications in DLBCL.
Aims: To analyze the phenotypic and functional profile of peripheral blood T lymphocyte compartment in DLBCL patients at diagnosis, and to assess the long-term impact of R-CHOP chemoimmunotherapy on T cell populations and functional competence.
Patients and Methods: We prospectively compared 32 consecutive newly diagnosed DLBCL patients with 27 healthy, age- and sex-matched controls for: 1) absolute number (/mcl) and percentage (over PBMC) of the main T cell subsets: "conventional CD4+ and CD8+, double positive (CD4+CD8+), double negative (CD4-CD8-), CD56+ innate-like, and FOXP3+CD25bright regulatory T cells (Treg), measured by blood cell count and multi-parameter flow cytometric (FACS) analysis; 2) functional capability of T cell subsets, evaluated as the frequency of IFN gamma (IFNg)-producing cells upon a short-term (6-hr) stimulation with PMA/ionomycin, and the percentage of GrzB+ (cytotoxic granule marker) cells; and 3) plasma concentration of selected cytokines. Patients were analyzed at diagnosis, during and at different timepoints after chemoimmunotherapy completion.
Results: DLBCL patients at diagnosis showed lower lymphocyte count (p<.001), mostly due to a selective decrement of CD4+ T cells (p=.003) and B lymphocytes (p=.0001). The absolute number of CD8+ or innate-like CD56+ T cells were not affected. As a result, the relative CD4/CD8 ratio was decreased (p=.009). The absolute number, but not the percentage, of Treg was slightly reduced with respect to controls. While CD8+ and innate-like T cell subsets transiently decreased during R-CHOP cycles, CD4+ T cell and Treg absolute numbers remained significantly lower than controls up to 1 year after therapy completion. As a result, CD4/CD8 ratio also remained lower than controls, up to 1 year after therapy.
The phenotypically skewed T cell profile of DLBCL patients at diagnosis was associated with severe functional alterations, as the frequencies of IFNg-producing and Granzyme B-expressing cells were increased in both CD4+ (p=.007 for IFNg+ and p=.005 for GrzB+) and CD8+ (p=.002 for IFNg+ and p<.0001 for GrzB+) conventional T cell subsets. Noteworthy, the functional competence of innate-like CD56+ T cells showed no difference with controls. The frequency of GrzB+ and IFNg-producing CD4+ and CD8+ T cells transiently decreased during therapy, thus becoming comparable to controls, raised again by three months after therapy, and was persistingly higher than controls up to 1 year after therapy completion.
Finally, IL-6 and IL-10 plasma levels were significantly elevated in DLBCL at diagnosis (p<.0001 and p=.011, respectively), and gradually normalized, shortly after R-CHOP therapy completion.
Interestingly, CD4+ and CD8+ T cell capability to promptly produce IFNg was significantly higher in GC, but not in non-GC DLBCL patients at diagnosis, suggesting that patients T cell functional features may depend on DLBCL phenotype. Moreover, a higher percentage of IFNg+ T cells was observed in patients attaining a long-term remission, but not in resistant or relapsing ones, hinting to the association of immune competence with therapy outcome.
Conclusions: T cell-dependent memory and effector capabilities might significantly contribute to the success of chemoimmunotherapy strategies, as suggested by the recently discovered vaccinal effect. The novel information provided by this study may reveal useful for innovative therapeutic approaches more focused on exploiting the dynamics of host immune cells in fighting cancer.
*MC Cox and S Battella contributed equally to this work

Disclosures
No relevant conflicts of interest to declare.