T cells targeting a neuronal paraneoplastic antigen mediate tumor rejection and trigger CNS autoimmunity with humoral activation

Abstract

Paraneoplastic neurologic diseases (PND) involving immune responses directed toward intracellular antigens are poorly understood. Here, we examine immunity to the PND antigen Nova2, which is expressed exclusively in central nervous system (CNS) neurons. We hypothesized that ectopic expression of neuronal antigen in the periphery could incite PND. In our C57BL/6 mouse model, CNS antigen expression limits antigen-specific CD4+ and CD8+ T-cell expansion. Chimera experiments demonstrate that this tolerance is mediated by antigen expression in nonhematopoietic cells. CNS antigen expression does not limit tumor rejection by adoptively transferred transgenic T cells but does limit the generation of a memory population that can be expanded upon secondary challenge in vivo. Despite mediating cancer rejection, adoptively transferred transgenic T cells do not lead to paraneoplastic neuronal targeting. Preliminary experiments suggest an additional requirement for humoral activation to induce CNS autoimmunity. This work provides evidence that the requirements for cancer immunity and neuronal autoimmunity are uncoupled. Since humoral immunity was not required for tumor rejection, B-cell targeting therapy, such as rituximab, may be a rational treatment option for PND that does not hamper tumor immunity.

Keywords: Autoimmunity; Cancer; Cellular immunity; Humoral immunity; Paraneoplastic neurologic disease.

Figure 1. Selective Expression of b-galactosidase in N2-LacZ mice

(A) Schematic diagram of the breeding strategy for N2-LacZ mice. Nova2-Cre-β-actin-LacZ (N2-LacZ) mice are double transgenic F1 offspring of crossing Nova2-Cre transgenic mice with chicken β-actin-LacZ transgenic mice. Upon induction of Cre activity in β-actin-LacZ X Nova2-Cre mice, the loxP-flanked STOP sequence is removed and LacZ is expressed in neurons expressing Nova2. (B) X-gal staining of WT, N2-Cre and N2-LacZ mouse brains. (C) qPCR analysis of LacZ mRNA in WT and N2-LacZ mouse organs normalized to the housekeeping gene, β-actin. Presented are the fold changes of LacZ expression in N2-LacZ mouse organs relative to the same tissue in littermate control mice. Data shown is mean+/−SD and is representative of three experiments. (D) b-gal staining by immunohistochemistry of organs of N2-LacZ and wild type mice. Arrows indicate b-gal expression (brown) in neurons. Magnification 600×: Bar indicates 20 µm. Hemotoxylin was used as a counterstain.

Figure 2. Testing of Humoral and Cellular tolerance to b-galactosidase in N2-LacZ mice

(A) Western blots of serum from N2-LacZ or Littermate control mice immunized with b-gal/CFA and PTx. Relative density was calculated by normalizing to a known commercial b-gal monoclonal antibody. (B–F) N2-LacZ or Littermate control mice were immunized with AdV-b-gal and PTx. (B) IFNγ ELISPOT responses of splenic CD4+ T cells cultured with irradiated and peptide pulsed splenocytes 13 days after immunization. (C) Representative FACS plots of CD8+ and tetramer positive cells 22 days after immunization. Gating strategy presented in Supporting information Fig. 3A. (D) Summary of (C), 8 mice per group. (E) IFNγ ELISPOT responses of splenic CD8+ T cells cultured with EL4 cells pulsed with 1 µM ova (irrelevant), b-gal p96, b-gal p497, or E22 cells, 13 days after immunization. (F) IFNγ ELISPOT responses of splenic CD8+ T cells to EL4 cells pulsed with decreasing amounts of b-gal p96 or p497 peptides 13 days post immunization. No IFNγ spots were detected from N2-LacZ mice at any p96 peptide concentration indicated by . Data are presented as mean+/−SD of three mice per group unless otherwise indicated. All data shown is representative of at least 3 experiments. NS indicates difference is not statistically significant by 2 tailed t-test. * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.001. Mice exhibited no signs of ataxia, hunched posturing or death.

Figure 3. N2-lacZ mice respond to in vivo b-gal challenge in the periphery

(A) Percent cytotoxicity of peptide pulsed CFSE labeled splenocytes infused 13 days after AdV-b-gal and PTx immunization. Percent cytotoxicity was calculated by comparing survival of CFSE labeled cells in immunized mice with non-immunized controls. Data presented is from one of five representative mice. Gating strategy is presented in Supporting information Fig. 3C (B) Tumor diameter of N2-LacZ (blue) or littermate (red) mice (5 per group) immunized with AdV-b-gal and PTx and challenged with b-gal expressing WP4 cells 10 days later. Data presented are mean+/−SD. *** indicates p<0.001 by repeated measure two-way ANOVA. All data is representative of at least 3 experiments.

Figure 4. T cell tolerance to b-gal in N2-LacZ mice is not due to b-gal expression in peripheral radio-resistant cells or in hematopoietic cells

Lethally irradiated WT or N2-LacZ host mice were reconstituted with BG1 and BG2 bone marrow. Reconstitution was assessed by measuring (A) %Vβ7+ CD8+ T cells, (B) %Vα11+ CD4+ T cells and (C) %CD25+/Foxp3+ of Vα11+ and Vα11- fractions. Gating strategy presented in Supporting information Fig. 3B. (D) %p96 tetramer+ CD8+ T cells in lethally irradiated WT or N2-LacZ host mice reconstituted with a mix of BG1 and WT or BG1 and N2-LacZ bone marrow and challenged with AdV-b-gal and PTx and 13 days later (n=6 mice per group, mean+SD). All data shown is representative of at least 3 experiments. NS indicates difference is not statistically significant by 2-tailed t-test. * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.001. Mice exhibited no signs of ataxia, hunched posturing or death.

Figure 5. BG1 and BG2 T cells reject tumor but do not cause autoimmune brain disease

(A) Experimental design: 5 million CD8+ +/− 5 million CD4+ cells from BG1, BG2 or WT mice were transferred into WT or N2-LacZ hosts and challenged with b-gal expressing WP4 cells. 30 days later, mice were secondarily challenged with AdV-b-gal and PTx. (B) Tumor growth in N2-LacZ or WT hosts that received T cell transfer. (C) Circulating b-gal p96 tetramer+ cells and (D) Western blot of serum b-gal antibody 28 days post tumor challenge (primary challenge). (E) % b-gal p96 or ova tetramer+ CD8+ T cells 8 days after AdV-b-gal and PTx (secondary challenge). (F) b-gal p96 specific CD8+ Vβ7⁺ CD3 cells were assessed for markers of effector (CD62L^lo, CD44^hi), central memory (CD62L^hi, CD4+4^hi) and naïve T cells (CD62L⁺, CD44^lo and CD44^int) 10 days after secondary challenge (n=8 mice per group, mean+SD). Gating strategy for C–F is presented in Supporting information Fig. 3A and B. *** indicates p<0.001 by 2-tailed t-test. Data is representative of two experiments.

Figure 6. T cells and B cells collaborate to generate neuronal targeting

Perfused brain sections were stained with hematoxylin and eosin and TUNEL. (A) Cerebral cortex. (B) Hippocampus. In A and B, arrows indicate dying neurons in the dentate gyrus. Arrowheads indicate normal reference neurons. Magnification 600×: Bar indicates 20 µm. (C) Intraparenchymal blood vessel. Arrows indicate neutrophilic infiltrate.