T cell-selective deletion of Oct1 protects animals from autoimmune neuroinflammation while maintaining neurotropic pathogen response

Abstract

Background: Treatments for autoimmune diseases aim to dampen autoreactivity while preserving normal immune function. In CD4⁺ T cells, the transcription factor Oct1/Pou2f1 is a dispensable transcription factor for T cell development and response to primary infection, but promotes expression of target genes, including Il2 and Ifng, under conditions of antigen reencounter. As a result, they are more strongly expressed upon secondary stimulation. Such repeated antigen encounters occur in memory recall responses, in autoimmunity where self-antigen can be recognized multiple times, and in chronic infection where foreign antigen is persistent. Based on these previous findings, we hypothesized that Oct1 loss would protect animals from autoimmunity but maintain normal responses to pathogens in the CNS.

Objective: We used a conditional mouse Oct1 (Pou2f1) allele and a CD4-Cre driver to determine the effect of T cell-specific Oct1 loss on autoimmune- and viral-induced neuroinflammation using an autoantigen-driven EAE model of autoimmunity and a JHMV model of viral infection.

Results: Oct1 conditional deletion mitigated clinical scores and reduced infiltrating T cells and cytokine production in the EAE model. Consistently, Oct1-deficient CD4⁺ T cells stimulated in vitro showed increased expression of markers associated with T cell anergy, particularly in the absence of co-stimulatory signals. In contrast, anti-viral T cell effector functions are intact in the absence of Oct1, with no changes in neuroinflammation, infiltrating T cells or cytokine production.

Conclusion: Our findings uncover a significant difference between the effect of Oct1 loss on autoimmune and anti-pathogen responses, which potentially could be exploited for therapeutic benefit.

Keywords: Experimental autoimmune encephalomyelitis; JHMV; Oct1/POU2F1; T lymphocytes.

Fig. 1

Loss of Oct1 in T cells protects mice using an EAE model of MS. a CD4-Cre;Oct1^fl/fl (n = 9) or Oct1^fl/fl (n = 10) mice were injected with MOG_35–55 peptide and pertussis toxin to generate EAE. Clinical scores were determined during the post-treatment timecourse. b Representative LFB staining of thoracic spinal cord sections from animals taken at peak disease (day 21). Areas of demyelination are outlined in red. c Quantification of demyelination in experimental mice. Mean % demyelination from six sections of two mice. d Cervical lymph node lymphocytes were isolated from EAE-induced CD4-Cre;Oct1^fl/fl (n = 6) or Oct1^fl/fl (n = 6) mice and analyzed by flow cytometry. Frequencies of CD4 and CD8 cells from representative animals are shown. e Mean CD4⁺ and CD8⁺ T cell percentages (left panel) and total cell numbers (right panel). Cells were independently purified from the CLNs of six separate six mice. f Representative data showing frequencies of cytokine-producing CD4⁺ cells in the CLN. g Percentages (left and middle panels) or total cell numbers (right panel) of cytokine-producing CD4⁺ T cells are plotted. N = 6 for each group. Mean of results is shown. h Mean CD4⁺ and CD8⁺ T cell percentages in the spinal cords. N = 3–4 for each group. i Cytokine-producing CD4⁺ T cell percentages (left panel) and total cell numbers (right panel). Cells were independently purified from the spinal cords of separate six mice

Fig. 2

In vitro stimulation of T cells lacking Oct1 results in decreased expression of markers associated with activation and increased expression of markers associated with anergy. a Oct1-deficient and control CD4⁺ T cells stimulated in vitro with indicated antibodies and analyzed by flow cytometry. Representative frequencies of ICOS-expressing CD4⁺ CD44⁺ cells are shown. b Quantification of cells independently purified from the spleens of three mice, with three technical culture replicates for each mouse. c Representative flow cytometry plots showing frequencies of CD25-expressing CD4⁺ CD44⁺ cells in Oct1-deficient and control CD4⁺ T cells. d Quantification from three animals. e Representative expression of CTLA-4 in CD4⁺ CD44⁺ T cells is plotted as histograms for Oct1-deficient and control CD4⁺ T cells. f CTLA4⁺ percentages from three animals, with three culture replicates per animal, are plotted. g Expression of FR4 and CD73 in Oct1-deficient and control CD4⁺CD44⁺ cells. h Averaged percentages of FR4^hiCD73^hiCD4⁺CD44⁺ cells are plotted

Fig. 3

The absence of Oct1 in T cells does not impact disease in JHMV-infected mice. a CD4-Cre;Oct1^fl/fl (n = 15) or Oct1^fl/fl mice (n = 14) were infected i.c. with 200 PFU of JHMV and disease severity assessed. Clinical disease was recorded to day 21 p.i. b Brain viral titers were determined at days 7 and 21 p.i., (n.d., not detected). c Representative LFB stained thoracic spinal cord sections from experimental mice at day 12 p.i. d Quantification of average demyelination from CD4-Cre;Oct1^fl/fl (n = 4, 12 dpi; n = 3, 21dpi) and Oct1^fl/fl mice (n = 3, 12 dpi; n = 5, 12 dpi) at days 12 and 21 p.i

Fig. 4

Normal immune responses in Oct1 T cell-deficient mice during JHMV infection. a CD4-Cre;Oct1^fl/fl or Oct1^fl/fl mice were infected i.c. with 200 PFU of JHMV and sacrificed at days 7 (n = 8), 12 (n = 4–5), and 21 (n = 6) p.i. to assess T cell infiltration into the brain. Representative flow analysis depicting CD4⁺ T cell infiltration into brains of mice at day 7 p.i. b Quantification of CD4⁺ T cells as shown by calculating both frequencies and numbers of isolated cells. c Representative flow analysis depicting CD8⁺ T cell infiltration into brains of mice at day 7 p.i. d Quantification of CD8⁺ T cells as shown by calculating both frequencies and numbers of isolated cells. e Representative M133-147 tetramer staining of CD4⁺ T cells from brains of JHMV-infected experimental mice. f Quantification of frequency and numbers of M133-147 tetramer CD4⁺ T cells from experimental groups. g Representative S510-518 tetramer staining of CD8⁺ T cells from brains of JHMV-infected experimental mice. h Quantification of frequency and numbers of M133-147 tetramer CD4⁺ T cells from experimental groups. Data presented are derived from two independent experiments; day 7 p.i., CD4-Cre;Oct1^fl/fl n = 8,Oct1^fl/fl mice n = 8; day 12 p.i., CD4-Cre;Oct1^fl/fl n = 5,Oct1^fl/fl mice n = 4. i IFNγ-producing CD4⁺ (left panel) and CD8⁺ (right panel) CNS-infiltrating T cell percentages are shown for representative animals. j Averaged frequencies (left panel) and total cell numbers (right panel) of CD4⁺ and CD8⁺ cells analyzed as in b. N = 6 for each group