Skip to main content

CBD Suppression of EAE Is Correlated with Early Inhibition of Splenic IFN-γ + CD8+ T Cells and Modest Inhibition of Neuroinflammation


In this study cannabidiol (CBD) was administered orally to determine its effects and mechanisms in the experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis (MS). We hypothesized that 75 mg/kg of oral CBD given for 5 days after initiation of disease would reduce EAE severity through suppression of either the early peripheral immune or late neuroimmune response. EAE was induced in C57BL/6 mice at two different magnitudes, and peripheral inflammatory and neuroinflammatory responses were measured at days 3, 10, and 18. Th1, Th17, Tc1, Tc17, Tregs, and myeloid derived suppressor cells (MDSC) were identified from the lymph nodes and spleens of each mouse to determine if CBD altered the suppressor cell or inflammatory cell populations in secondary lymphoid tissues. Additionally, neuroinflammation was identified in brain and spinal cord tissues using various immunohistochemical techniques and flow cytometry. Early treatment of EAE with oral CBD reduced clinical disease at the day 18 timepoint which correlated with a significant decrease in the percentage of MOG35–55 specific IFN-γ producing CD8+ T cells in the spleen at day 10. Analysis of both T cell infiltration and lesion size within the spinal cord also showed a moderate reduction in neuroinflammation within the central nervous system (CNS). These results provide evidence that oral CBD suppressed the peripheral immune response that precedes neuroinflammation; however, analysis of the neuroinflammatory endpoints also suggest that the modest reduction in neuroinflammation was only partially responsible for CBD’s neuroprotective capability.

CBD was administered orally for the first 5 days following initiation of EAE. CBD attenuated clinical disease, and we found that CBD suppressed IFN-γ producing CD8+ T cells in the spleen at day 10. There was also modest suppression of neuroinflammation. Together these data demonstrate that early, oral administration of CBD protected mice from disease, but the modest effects on neuroinflammation suggest other mechanisms participate in CBD’s neuroprotective effect in EAE.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Fig. 9





Bovine serum albumin




Cannabinoid receptors


Corn oil




Experimental Autoimmune Encephalomyelitis


Fixable Viability Dye

FC buffer:

Flow cytometry buffer


Glial Fibrillary Acidic Protein


Heat-killed Mycobacterium tuberculosis H37Ra


Hematoxylin and eosin


Inflammatory bowel disease




Multiple Sclerosis


Myelin basic protein


Myelin oligodendrocyte protein


Myeloid derived suppressor cell


Neutral buffered formalin


PBS containing 0.05% Tween 20


Pertussis toxin


Phorbol 12-myristate-13-acetate and ionomycin


Phosphate buffered saline


Proteolipid protein


Rheumatoid arthritis


Theiler’s murine encephalomyelitis virus


Triton X-100


Vascular cell-adhesion molecule-1


  • Annunziato F, Cosmi L, Santarlasci V, Maggi L, Liotta F, Mazzinghi B, Parente E, Fili L, Ferri S, Frosali F, Giudici F, Romagnani P, Parronchi P, Tonelli F, Maggi E, Romagnani S (2007) Phenotypic and functional features of human Th17 cells. J Exp Med 204:1849–1861

    Article  CAS  Google Scholar 

  • Babbe H, Roers A, Waisman A, Lassmann H, Goebels N, Hohlfeld R, Friese M, Schroder R, Deckert M, Schmidt S, Ravid R, Rajewsky K (2000) Clonal expansions of CD8(+) T cells dominate the T cell infiltrate in active multiple sclerosis lesions as shown by micromanipulation and single cell polymerase chain reaction. J Exp Med 192:393–404

    Article  CAS  Google Scholar 

  • Bogie JF, Stinissen P, Hendriks JJ (2014) Macrophage subsets and microglia in multiple sclerosis. Acta Neuropathol 128:191–213

    Article  CAS  Google Scholar 

  • Bongioanni P, Lombardo F, Moscato G, Mosti S, Meucci G (1999) T-cell interferon gamma receptor binding in interferon beta-1b-treated patients with multiple sclerosis. Arch Neurol 56:217–222

    Article  CAS  Google Scholar 

  • Boven LA, Van Meurs M, Van Zwam M, Wierenga-Wolf A, Hintzen RQ, Boot RG, Aerts JM, Amor S, Nieuwenhuis EE, Laman JD (2006) Myelin-laden macrophages are anti-inflammatory, consistent with foam cells in multiple sclerosis. Brain 129:517–526

    Article  Google Scholar 

  • Brucklacher-Waldert V, Stuerner K, Kolster M, Wolthausen J, Tolosa E (2009) Phenotypical and functional characterization of T helper 17 cells in multiple sclerosis. Brain 132:3329–3341

    Article  Google Scholar 

  • Burstein S (2015) Cannabidiol (CBD) and its analogs: a review of their effects on inflammation. Bioorg Med Chem 23:1377–1385

    Article  CAS  Google Scholar 

  • Crook KR, Liu P (2014) Role of myeloid-derived suppressor cells in autoimmune disease. World J Immunol 4:26–33

    Article  Google Scholar 

  • Dhital S, Stokes JV, Park N, Seo KS, Kaplan BL (2017) Cannabidiol (CBD) induces functional Tregs in response to low-level T cell activation. Cell Immunol 312:25–34

    Article  CAS  Google Scholar 

  • Elliott DM, Singh N, Nagarkatti M, Nagarkatti PS (2018) Cannabidiol attenuates experimental autoimmune encephalomyelitis model of multiple sclerosis through induction of myeloid-derived suppressor cells. Front Immunol 9:1782

    Article  Google Scholar 

  • Feger U, Luther C, Poeschel S, Melms A, Tolosa E, Wiendl H (2007) Increased frequency of CD4+ CD25+ regulatory T cells in the cerebrospinal fluid but not in the blood of multiple sclerosis patients. Clin Exp Immunol 147:412–418

    Article  CAS  Google Scholar 

  • Fletcher JM, Lalor SJ, Sweeney CM, Tubridy N, Mills KH (2010) T cells in multiple sclerosis and experimental autoimmune encephalomyelitis. Clin Exp Immunol 162:1–11

    Article  CAS  Google Scholar 

  • Giacoppo S, Pollastro F, Grassi G, Bramanti P, Mazzon E (2017) Target regulation of PI3K/Akt/mTOR pathway by cannabidiol in treatment of experimental multiple sclerosis. Fitoterapia 116:77–84

    Article  CAS  Google Scholar 

  • Gravano DM, Hoyer KK (2013) Promotion and prevention of autoimmune disease by CD8+ T cells. J Autoimmun 45:68–79

    Article  CAS  Google Scholar 

  • Haas J, Hug A, Viehover A, Fritzsching B, Falk CS, Filser A, Vetter T, Milkova L, Korporal M, Fritz B, Storch-Hagenlocher B, Krammer PH, Suri-Payer E, Wildemann B (2005) Reduced suppressive effect of CD4+CD25high regulatory T cells on the T cell immune response against myelin oligodendrocyte glycoprotein in patients with multiple sclerosis. Eur J Immunol 35:3343–3352

    Article  CAS  Google Scholar 

  • Hegde VL, Nagarkatti PS, Nagarkatti M (2011) Role of myeloid-derived suppressor cells in amelioration of experimental autoimmune hepatitis following activation of TRPV1 receptors by cannabidiol. PLoS One 6:e18281

    Article  CAS  Google Scholar 

  • Hegde VL, Singh UP, Nagarkatti PS, Nagarkatti M (2015) Critical role of mast cells and peroxisome proliferator-activated receptor gamma in the induction of myeloid-derived suppressor cells by marijuana Cannabidiol in vivo. J Immunol 194:5211–5222

    Article  CAS  Google Scholar 

  • Huseby ES, Liggitt D, Brabb T, Schnabel B, Ohlen C, Goverman J (2001) A pathogenic role for myelin-specific CD8(+) T cells in a model for multiple sclerosis. J Exp Med 194:669–676

    Article  CAS  Google Scholar 

  • Ioannou M, Alissafi T, Lazaridis I, Deraos G, Matsoukas J, Gravanis A, Mastorodemos V, Plaitakis A, Sharpe A, Boumpas D, Verginis P (2012) Crucial role of granulocytic myeloid-derived suppressor cells in the regulation of central nervous system autoimmune disease. J Immunol 188:1136–1146

    Article  CAS  Google Scholar 

  • Kaplan BL, Rockwell CE, Kaminski NE (2003) Evidence for cannabinoid receptor-dependent and -independent mechanisms of action in leukocytes. J Pharmacol Exp Ther 306:1077–1085

    Article  CAS  Google Scholar 

  • Kaplan BL, Springs AE, Kaminski NE (2008) The profile of immune modulation by cannabidiol (CBD) involves deregulation of nuclear factor of activated T cells (NFAT). Biochem Pharmacol 76:726–737

    Article  CAS  Google Scholar 

  • Karmaus PW, Wagner JG, Harkema JR, Kaminski NE, Kaplan BL (2013) Cannabidiol (CBD) enhances lipopolysaccharide (LPS)-induced pulmonary inflammation in C57BL/6 mice. J Immunotoxicol 10:321–328

    Article  CAS  Google Scholar 

  • Kozela E, Lev N, Kaushansky N, Eilam R, Rimmerman N, Levy R, Ben-Nun A, Juknat A, Vogel Z (2011) Cannabidiol inhibits pathogenic T cells, decreases spinal microglial activation and ameliorates multiple sclerosis-like disease in C57BL/6 mice. Br J Pharmacol 163:1507–1519

    Article  CAS  Google Scholar 

  • Kozela E, Juknat A, Kaushansky N, Rimmerman N, Ben-Nun A, Vogel Z (2013) Cannabinoids decrease the th17 inflammatory autoimmune phenotype. J NeuroImmune Pharmacol 8:1265–1276

    Article  Google Scholar 

  • Langrish CL, Chen Y, Blumenschein WM, Mattson J, Basham B, Sedgwick JD, McClanahan T, Kastelein RA, Cua DJ (2005) IL-23 drives a pathogenic T cell population that induces autoimmune inflammation. J Exp Med 201:233–240

    Article  CAS  Google Scholar 

  • Li R, Rezk A, Healy LM, Muirhead G, Prat A, Gommerman JL, Bar-Or A, Team MCBciM (2015) Cytokine-defined B cell responses as therapeutic targets in multiple sclerosis. Front Immunol 6:626

    Article  Google Scholar 

  • M. Mecha AF, F.J. Carrillo-Salinas, C. Guaza (2017) Chapter 93: Cannabidiol and Multiple Sclerosis. In: Handbook of Cannabis and Related Pathologies (Preedy VR, ed), p 1 online resource. S.l.: Academic Press

  • Malfait AM, Gallily R, Sumariwalla PF, Malik AS, Andreakos E, Mechoulam R, Feldmann M (2000) The nonpsychoactive cannabis constituent cannabidiol is an oral anti-arthritic therapeutic in murine collagen-induced arthritis. Proc Natl Acad Sci U S A 97:9561–9566

    Article  CAS  Google Scholar 

  • Mallucci G, Peruzzotti-Jametti L, Bernstock JD, Pluchino S (2015) The role of immune cells, glia and neurons in white and gray matter pathology in multiple sclerosis. Prog Neurobiol 127-128:1–22

    Article  CAS  Google Scholar 

  • Mangmool S, Kurose H (2011) G(i/o) protein-dependent and -independent actions of pertussis toxin (PTX). Toxins (Basel) 3:884–899

    Article  CAS  Google Scholar 

  • Matsuda LA, Lolait SJ, Brownstein MJ, Young AC, Bonner TI (1990) Structure of a cannabinoid receptor and functional expression of the cloned cDNA. Nature 346:561–564

    Article  CAS  Google Scholar 

  • Mecha M, Feliu A, Inigo PM, Mestre L, Carrillo-Salinas FJ, Guaza C (2013) Cannabidiol provides long-lasting protection against the deleterious effects of inflammation in a viral model of multiple sclerosis: a role for A2A receptors. Neurobiol Dis 59:141–150

    Article  CAS  Google Scholar 

  • Miller SD, Karpus WJ (1994) The immunopathogenesis and regulation of T-cell-mediated demyelinating diseases. Immunol Today 15:356–361

    Article  CAS  Google Scholar 

  • Moline-Velazquez V, Cuervo H, Vila-Del Sol V, Ortega MC, Clemente D, de Castro F (2011) Myeloid-derived suppressor cells limit the inflammation by promoting T lymphocyte apoptosis in the spinal cord of a murine model of multiple sclerosis. Brain Pathol 21:678–691

    Article  CAS  Google Scholar 

  • Munro S, Thomas KL, Abu-Shaar M (1993) Molecular characterization of a peripheral receptor for cannabinoids. Nature 365:61–65

    Article  CAS  Google Scholar 

  • Nichols JM, Kaplan BL (2020) Immune responses regulated by cannabidiol. Cannabis Cannabinoid Res 5:12–31

  • Pagany M, Jagodic M, Schubart A, Pham-Dinh D, Bachelin C, Baron van Evercooren A, Lachapelle F, Olsson T, Linington C (2003) Myelin oligodendrocyte glycoprotein is expressed in the peripheral nervous system of rodents and primates. Neurosci Lett 350:165–168

    Article  CAS  Google Scholar 

  • Rao P, Segal BM (2012) Experimental autoimmune encephalomyelitis. Methods Mol Biol 900:363–380

    Article  CAS  Google Scholar 

  • Sallusto F, Impellizzieri D, Basso C, Laroni A, Uccelli A, Lanzavecchia A, Engelhardt B (2012) T-cell trafficking in the central nervous system. Immunol Rev 248:216–227

    Article  Google Scholar 

  • Sinha S, Boyden AW, Itani FR, Crawford MP, Karandikar NJ (2015) CD8(+) T-cells as immune regulators of multiple sclerosis. Front Immunol 6:619

    Article  Google Scholar 

  • Tzartos JS, Friese MA, Craner MJ, Palace J, Newcombe J, Esiri MM, Fugger L (2008) Interleukin-17 production in central nervous system-infiltrating T cells and glial cells is associated with active disease in multiple sclerosis. Am J Pathol 172:146–155

    Article  CAS  Google Scholar 

  • Vos CM, Geurts JJ, Montagne L, van Haastert ES, Bo L, van der Valk P, Barkhof F, de Vries HE (2005) Blood-brain barrier alterations in both focal and diffuse abnormalities on postmortem MRI in multiple sclerosis. Neurobiol Dis 20:953–960

    Article  CAS  Google Scholar 

Download references


The authors thank Dr. T. Graham Rosser for help with illustrations. Funding was provided by National Institutes of Health training grant T35OD010432, Center of Biomedical Research Excellence (COBRE) grant P20GM103646 and Institutional Development Award (IDeA) grant P20GM103476.

Author information

Authors and Affiliations


Corresponding author

Correspondence to Barbara L. F. Kaplan.

Ethics declarations

Experiments were approved by Mississippi State University Institutional Animal Care and Use Committee (IACUC).

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Conflict of Interest

The authors declare that they have no competing interests.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

Supplemental Fig. 1

Gating strategy for identifying Tc1, Tc17, Th1, and Th17cells. CD8+IFN-γ+, CD8+IL17A+, CD4+IFN-γ+, CD4+IL-17A+ lymphocytes were identified in the live singlet population. (PNG 1007 kb)

High Resolution (TIF 6997 kb)

Supplemental Fig. 2

Cell counts of inflammatory T cells from ex vivo restimulated splenocytes. These cell counts correspond to the percentages of inflammatory T cells given in Fig. 3 of the main body of the paper. * p < 0.05 differences between SAL/CO and EAE/CO; ‡ p < 0.05 difference between SAL/CO and Mild EAE/CO; # p < 0.05 difference between EAE/CO and EAE/CBD; @p < 0.05 difference between EAE/CO and Mild EAE/CO € p < 0.05 difference between EAE/CBD and Mild EAE/CBD. (PNG 305 kb)

High Resolution (TIF 3174 kb)

Supplemental Fig. 3

Cell counts of inflammatory T cells from ex vivo restimulated cells isolated from lymph nodes. These cell counts correspond to the percentages of inflammatory T cells given in Fig. 4 of the main body of the paper. * p < 0.05 differences between SAL/CO and EAE/CO; ‡ p < 0.05 difference between SAL/CO and Mild EAE/CO; § p < 0.05 difference between Mild EAE/CO and Mild EAE/CBD. (PNG 375 kb)

High Resolution (TIF 5506 kb)

Supplemental Fig. 4

Gating strategy for identifying MDSCs and Tregs. Tregs were identified as CD4+CD25+FoxP3+ Lymphocytes in the singlet population. Granulocytic MDSC are identified as CD11b+Ly6CloLy6G+ cells and monocytic MDSC are identified as CD11b+Ly6C+Ly6G cells in the singlet population. (PNG 1288 kb)

High Resolution (TIF 8696 kb)

Supplemental Fig. 5

Cell counts of regulatory cells from secondary lymphoid organs. These cell counts correspond to the percentages of regulator cells given in Fig. 2 of the main body of the paper. * p < 0.05 differences between SAL/CO and EAE/CO; ‡ p < 0.05 difference between SAL/CO and Mild EAE/CO; # p < 0.05 difference between EAE/CO and EAE/CBD; @p < 0.05 difference between EAE/CO and Mild EAE/CO € p < 0.05 difference between EAE/CBD and Mild EAE/CBD. (PNG 409 kb)

High Resolution (TIF 4316 kb)

Supplemental Fig. 6

Cell counts of T cells isolated from the spinal cord of EAE mice. These cell counts correspond to the percentages of T cells given in Fig. 9D of the main body of the paper. (PNG 236 kb)

High Resolution (TIF 2586 kb)

Rights and permissions

Reprints and Permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Nichols, J.M., Kummari, E., Sherman, J. et al. CBD Suppression of EAE Is Correlated with Early Inhibition of Splenic IFN-γ + CD8+ T Cells and Modest Inhibition of Neuroinflammation. J Neuroimmune Pharmacol 16, 346–362 (2021).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: