CD4+ NKT but not Conventional CD4+ T Cells Required for ACAID

Abstract

Abstract: : Purpose: CD4⁺ T cells are known to help CD8⁺ T effector cells develop. The role of CD4 ⁺ T cells in the development of CD8⁺ T regulatory (Tr) cells is not known. Previously we reported that the development of CD8⁺ Tr cells in ACAID is dependent on CD1d dependent invariant (i) (Vα14Jα18 – TCR) NKT cells. Since this populations of iNKT cells may be either double negative (DN, CD4^-CD8^-), or CD4⁺. The aim of this study was to determine if conventional CD4⁺ T or CD4⁺ iNKT cells were needed for development of efferent CD8⁺ Tr cells in ACAID. Methods: ACAID was induced in various wild type and knockout (KO) mice by inoculation of endotoxin free ovalbumin (OVA) in the anterior chamber (a.c.). Potential Tr cells were enriched from spleens by passing dissociated cells through Immulan® columns and tested by adding to a mixture of enriched T OVA effector cells and stimulator cells (thioglycolate induced OVA pulsed PECs) before adoptively transfering to an ear of a naïve mouse and measuring ear swelling a day later. Flow cytometry was used to determine the surface phenotype of lymphocyte subpopulations in the spleen post a.c. inocualtion. Results: Seven days post a.c. inoculation, CD4⁺ but not DN iNKT cells were increased in the spleens of ACAID mice. Intracellular staining showed that the CD4⁺ iNKT cells produced IL-10. The possibility for a role of conventional CD4⁺ T cells in the generation of Tr cells was ruled out since enriched T cells from spleens of OVA-a.c. inoculated Class II KO mice suppressed DTH in the LAT assay. However, when CD4⁺ cells were removed by in vivo treatment of WT, and ClassII KO mice with mAb to CD4 (GK1.5) neither were able to generate efferent Tr cells. Furthermore, reconstitution of Jα18 KO mice (lack iNKT cells) with CD4⁺ iNKT cells restored their ability to produce efferent Tr cells in ACAID. Conclusions: CD4⁺ iNKT cells but not conventional CD4⁺ T cells are required to generate CD8⁺ Tr cells in ACAID. Moreover, since CD8⁺ Tr cells are generated in the absence of Class II, Class II recognition is not required for CD1d dependent development of CD4⁺ iNKT cell population or their function in ACAID. The role of CD4 molecules on the iNKT cell population remains to be determined.

Keywords: ACAID • immune tolerance/privilege • immunomodulation/immunoregulation