A destructive feedback loop mediated by CXCL10 in central nervous system inflammatory disease

Abstract

Objective: Paraneoplastic neurologic disorders (PND) are autoimmune diseases associated with cancer and ectopic expression of a neuronal antigen in a peripheral tumor. Patients with PND harbor high-titer antibodies and T cells in their serum and cerebrospinal fluid (CSF) that are specific to the tumor antigen, and treatment with the immunosuppressant FK506 (tacrolimus) decreases CSF white blood cell counts. The objective of this study was to determine the effect of FK506 on CSF chemokine levels in PND patients.

Methods: CSF samples before and after FK506 treatment were tested by multiplex assay for the presence of 27 cytokines. Follow-up in vitro experiments aimed to determine whether T cells secrete CXCL10 in response to cognate antigen.

Results: Here we report that PND patients harbor high levels of the chemokine CXCL10 in their CSF. CXCL10 is a cytokine that recruits CXCR3(+) cells such as activated T cells, and we found that FK506 treatment specifically decreased CSF CXCL10 from among 27 cytokines tested. In vitro, CXCL10 was only produced during antigen-specific cognate interactions between T cells and antigen-presenting cells (APCs) when interferon-γ (IFNγ) receptors were present on the T cell.

Interpretation: These results support a model in which antigen-specific T cell stimulation by PND APCs triggers IFNγ, followed by CXCL10 production and further lymphocyte recruitment, suggesting that treatments targeting T cells or CXCL10 in the central nervous system (CNS) may interrupt a destructive positive feedback loop present in CNS inflammation.

Figure 1

CXCL10 is elevated in paraneoplastic neurologic disorder (PND) patient cerebrospinal fluid (CSF) and is decreased after FK506 treatment. (A) Levels of CXCL10 in the CSF of 8 PND patients with the Hu syndrome or paraneoplastic cerebellar degeneration (PCD) before and after FK506 treatment, as determined by CXCL10 ELISA. Bars represent the average of duplicate wells. p = 0.011 for patients with PND pre‐ versus post‐treatment by Wilcoxon signed rank test with p < 0.05 considered significant, indicating a decrease in CXCL10 with treatment. (B) Western blot of serum from PCD patient PCD0615 recognizing cdr2 (∼50kD). (C) CSF white blood cell (WBC) counts and CXCL10 levels over time in PND patient PCD0615, who was treated with repeated courses of FK506. Arrows indicate treatment periods.

Figure 2

Enrichment of CXCR3⁺ T cells in paraneoplastic neurologic disorder (PND) patient cerebrospinal fluid (CSF). (A) Flow cytometry analysis of PND patient cells showing CXCR3⁺CD4⁺ T cells (top panels) and CXCR3⁺CD8⁺ T cells (bottom panels) in CSF and peripheral blood. (B) Percentage of CD3⁺/CXCR3⁺ T cells in CSF and peripheral blood from individual PND patients. Percentage of CSF CD3⁺/CXCR3⁺ cells mean = 73; percentage of peripheral blood CD3⁺/CXCR3⁺ cells mean = 20.4. A t test assuming unequal variance was applied to the data with p < 0.05 considered significant. p = 2e10⁻⁸ for CSF versus peripheral blood CXCR3⁺ cells.

Figure 3

CXCL10 is produced in an antigen (Ag)‐specific fashion in cultures of human T cells and dendritic cells (DCs) from normal donors. (A) CXCL10 was measured by ELISA in supernatants from cocultures of CD8⁺ or CD4⁺ T cells and DCs presenting influenza antigen (Flu) or uninfected DCs (neg). (B) Interferon‐γ (IFNγ) Elispot assay of normal donor CD8⁺ and CD4⁺ T cells cultured with DCs presenting influenza antigen, performed in parallel with the experiment in A. Error bars represent standard deviation from the mean of triplicate wells for each condition.

Figure 4

T cell interferon‐γ (IFNγ) receptor is required for antigen (Ag)‐specific production of CXCL10 in cocultures of T cells and dendritic cells (DCs). (A–D) CXCL10 (A, B) and IFNγ (C, D) were measured in cocultures of T cells from influenza‐immunized CXCL10^−/− or wild‐type (wt) C57BL/6 mice with DC from CXCL10^−/− or wild type mice. For CD8⁺ cocultures (A, C), DCs were pulsed with influenza nucleoprotein peptide (Flu) or ovalbumin peptide control (neg). For CD4⁺ cocultures (B, D), DCs were fed apoptotic influenza‐infected 3T3 cells (Flu) or uninfected 3T3 cells (neg). (E) CXCL10 measured in cocultures set up as in A, using T cells from influenza‐immunized IFNγR^−/− mice. (F) IFNγ production was determined in parallel. Each experiment was repeated at least 3 times with similar results. CXCL10 in coculture supernatants was measured by ELISA and IFNg secretion was measured by Elispot.

Figure 5

Proposed model of the interactions leading to CXCL10 production by T cells and antigen‐presenting cells (APCs). The T cell is activated by its cognate antigen presented on an APC. The T cell secretes interferon‐γ (IFNγ) in response to the antigen and senses autocrine or paracrine IFNγ via the IFNγ receptor (IFNγR). The T cell and APC secrete CXCL10 in this antigen‐specific interaction, which recruits additional CXCR3⁺ cells into the area. The signal that tells the APC to produce CXCL10 was not investigated in the current experiments, but may be related to expression of the IFNγR by APCs or other signals.