Distinct Patterns of Systemic Immune Dysregulation in Indolent B Cell Lymphoma
Authors: Christopoulos Petros , Pfeifer Dietmar , Bartholomé Kilian , Follo Marie , Fisch Paul , Timmer Jens , Veelken Hendrik 
Published: October 2019

Abstract
Mutual interactions of the neoplastic clone with the non-neoplastic immune system may influence immune function and the clinical behaviour of lymphoma. Individuals with immunodeficiency or autoimmune diseases have an increased risk for lymphoma development. The immune microenvironment appears to have a major influence on the prognosis of indolent lymphomas. Conversely, leukemic lymphomas may also cause immunodeficiency: In CLL, direct lymphoma-T cell interactions, which may occur ubiquitously, induce defects in T cell functions (Görgün et al., 2005). We demonstrate here a systemic perturbance of cellular immunity in a prospective study in patients with untreated de novo, limited-stage, non-leukemic indolent B cell lymphomas. Calibrated, quantitative flow cytometry showed a significant reduction of circulating T helper (TH) cells in follicular (FL; n=11; p<0.005) and extranodal marginal zone (eMZL; n=7; p<0.05) lymphomas compared to age-matched healthy persons. Naive TH cells were strongest reduced to 51% (p=0.002) in FL and 24% (p=0.002) in eMZL. Regulatory T cells (CD4loCD25hi; CD4+FoxP3+) were affected less (p=0.04). T cell receptor excision circles within CD4+ cells as assessed by quantitative PCR were not altered in lymphoma patients, indicating neither increased increased thymic output nor homeostatic T cell proliferation to compensate the contracted pool of naive T cells. The TH memory compartments, the global numbers and subsets of CD8+ T (TC) cells, NK, and NKT cells were normal. The peripheral lymphocyte composition was altered differently in early CLL (stage Binet A; leukocyte counts < 28/nl; n=9) with increased TH (p=0.04) and TC (p=0.0002) cells. No significant changes in lymphocyte subsets were noted in monoclonal gammopathy of unknown significance (MGUS; n=6). The functional T cell phenotype in vivo was altered in eMZL as indicated by four- and twofold increased HLA-DR+ TH (p<0.02) and TC (p=0.05) cells. This T cell activation may also explain an increased fraction of terminally differentiated (CD45RA+CD27−) TC cells (p<0.05). Qualitatively similar abnormalities were seen in FL, where activated TH cells were more frequent (p<0.005), and in CLL, where activated TC cells were increased (p=0.04), but not in MGUS. Finally, an increased T cell activation may effect senescence, which was evident by elevated fractions of CD57+ and CD28− cells within the TC compartment of FL/eMZL (p<0.05) and CLL (p<0.005) patients. The activated T cell phenotype was paralleled by increased upregulation of activation markers (CD25, OX40, CD95, p<0.005 for each) and proliferation (p<0.005) by purified CD4 cells from FL/eMZL patients in a standardized anti-CD3/anti-CD28 stimulation culture. None of these parameters was significantly aberrant in CLL. Expression of the activation marker CD69, which is downregulated rapidly after T cell activation, was markedly reduced both in vivo and after in vitro stimulation in FL/eMZL. Collectively, these data demonstrate a global, “preactivated” and presenescent state of peripheral T cells in non-leukemic, indolent T cell lymphomas. Finally, a shift towards TH2 cells was evident in FL/eMZL TH stimulation cultures by increased secretion of IL-4 and IL-5 (p=0.01), but not of IL-2, IFNg, IL-10, and TNFa. This cytokine pattern was absent in CLL and MGUS. The TH2 shift, and the qualitative difference in the immune status in FL/eMZL versus CLL was validated by gene expression profiling of stimulated TH cells with Affymetrix U133 arrays. KEGG annotation revealed decreased expression of proximal TCR signalling molecules and TCR/CD28 transduction pathways with the exception of NFAT in FL/eMZL and CLL. Extensive correlative analyses between gene expression profiles and functional data indicated at least two distinct immune dysregulation patterns: A hyperreactivity/TH2 pattern which is operational even in early disease; and a B cell burden-dependent impairment of TCR signalling. The latter pattern predominates in CLL, which has a comparatively high B cell burden in early disease. These data are clinically relevant since we demonstrate in a prospective trial that untreated FL/eMZL patients fail to respond to protective hepatitis B vaccination (p<0.005). Precise definition of functional T cell defects will permit to study the causes, the prognostic influence, and potential reversibility of immune dysregulation patterns in indolent B cell lymphomas.