Characteristics of splenic CD8+ T cell exhaustion in patients with hepatitis C

Abstract

Summary There is increasing interest in the role of T cell exhaustion and it is well known that the natural history of chronic hepatitis C virus infection (HCV) is modulated by CD8+ T cell immunobiology. There are many pathways that alter the presence of exhaustive T cells and, in particular, they are functionally impaired by inhibitory receptors, such as programmed death-1 (PD-1) and T cell immunoglobulin and mucin domain-containing protein 3 (Tim-3). We obtained spleen, liver and peripheral blood (before and after splenectomy) lymphoid cells from 25 patients with HCV-related cirrhosis undergoing liver transplantation for end-stage disease or splenectomy for portal hypertension. In all samples we performed an extensive phenotypic study of exhaustion markers [PD-1, Tim-3, interferon (IFN)-γ) and their ligands (PD-L1, PD-L2, galectin-9] in CD8 + T cell subpopulations (both total and HCV-specific) and in antigen-presenting cells (APC; monocytes and dendritic cells). In the spleen, total and HCV-specific CD8+ T cells demonstrated enhanced markers of exhaustion, predominantly in the effector memory subpopulation. Similarly, splenic APC over-expressed inhibitory receptor ligands when compared to peripheral blood. Finally, when peripheral blood CD8 + T cells were compared before and after splenectomy, markers of exhaustion were reduced in splenic CD8 + T cells and APC. Our data in HCV-related cirrhosis suggest that CD8 + T cells in the spleen manifest a significantly higher exhaustion compared to peripheral blood and may thus contribute to the failure to control HCV. Counteracting this process may contribute to inducing an effective immune response to HCV.

Regulation of T cell responses by the receptor molecule Tim-3

“…Moreover, TCR-induced responses by Tim-3 + CD8 T cells were augmented by the addition of a soluble form of Tim-3 or Tim-3-specific antibodies, suggesting that Tim-3 enforces functional impairment. Following this report, several studies showed that the frequencies of Tim-3 + CD8 T cells were elevated in PBL from patients chronically infected with HBV or HCV and that these cells were in a dysfunctional state, with some of these studies providing evidence that impairment of CD8 T cell function was at least partly dependent on Tim-3 [76, 87, 86, 89, 99, 92]. …”

“…Studies employing mice have demonstrated that Tim-3 is essentially absent on naïve CD8 T cells but is expressed by CD8 T cells that become activated due to microbial infection [80, 37, 81-85]. In addition, a number of studies involving mice or human samples have shown that chronic infections result in high levels of Tim-3 expression by activated CD8 T cells [74, 76, 86, 87, 81, 88, 79, 89-92]. Lastly, data from mouse cancer models or analysis of patient samples revealed that CD8 T cells infiltrating tumors express Tim-3 [93-98].…”

Regulation of T cell responses by the surface receptor Tim-3

“…Tim-3 expression is largely absent on naïve CD8 T cells but is induced on CD8 T cells activated by acute microbial infections [56,170,174,[181][182][183][184]. It has also been shown that chronic infections in humans or mice lead to expression of Tim-3 on activated CD8 T cells [56,[185][186][187][188][189][190][191][192][193][194]. Lastly, Tim-3 expression has been demonstrated on tumor infiltrating lymphocyte (TIL) CD8 T cells from mouse cancer models as well as cancer patient samples [195][196][197][198][199][200].…”

“…Additionally, CD8 T cell responses were augmented by addition of soluble Tim-3 or Tim-3 antibody. Several reports that followed demonstrated that Tim-3 + CD8 T cells were elevated in HBV or HCV-infected patients and that these cells were also dysfunctional [185,188,189,191,193,201].…”

“…Spleen cells were resuspended in RPMI 1640 supplemented with 10% fetal bovine serum, 1% L-glutamine, 50 units/ml penicillin, 50 ug/ml streptomycin and 50 µM β-mercaptoethanol. OVA 257-264 peptide (1 µM) for CD8 T cells or LLO[190][191][192][193][194][195][196][197][198][199][200][201] (50 ng/ml) for CD4 T cells and GolgiPlug (Brefeldin A (BFA)) or GolgiStop (monensin) (BD Biosciences) were added to aliquots of cells followed by incubation for 5 hrs at 37˚C in a 5% CO 2 incubator. Cells were washed with stain buffer and incubated with antibodies for cell-surface markers.…”