A key to understanding MHC I's immunological activity has been to define the principles that govern its recognition by receptors in the adaptive and innate immune systems. To further understand MHC I's role in neurodevelopment, we sought to define the principles that govern MHC I recognition by neurons.
We found that picomolar levels of a self-MHC I molecule could inhibit retina neurite outgrowth in vitro. This neuroinhibitory activity was not due to toxic factors in the recombinant MHC I preparations because: 1) preabsorption with an anti-Db
, but not an anti-Kk
, mAb removed the inhibitory factor; 2) recombinant MHC I inhibited neurite outgrowth from syngeneic retinas, but had little effect on retinas from an allogeneic mouse strain; and 3) retinas from β2M−/−
mice were unaffected by recombinant MHC I. Recombinant MHC I's neuroinhibitory effect is unlikely to be mediated indirectly through infiltrating immune cells or glia cells in the E14 retinal explants because immune cells, such as dendritic cells, macrophages, and B cells, do not enter the retina until 1 wk after birth (24
), and T cells are still developing in the thymus at E14, and retina explants from MHC I-deficient mice were unaffected by recombinant MHC I. Although MHC I-deficient mice have deficiencies in CD8+
T cells, their other immune cells and retina glial cells are functional. This observation strongly argues against the notion that recombinant MHC I inhibited neurite outgrowth indirectly through infiltrating immune cells or glia. Finally, MHC I tetramer staining showed that MHC I receptor expression in the embryonic retina was limited to the RGCs and their precursor cells. The staining pattern was inconsistent with the staining of Müller glia, which arise well after E14 and have a distinctive laminar position, and microglia and astrocytes, which enter the eye after E16 and E19, respectively. Although we cannot completely rule out any contributions from immune cells or glia in our retina explant model, previous studies of immune cell development and our observations using retinas from two strains of MHC I-deficient mice, as well as MHC I tetramer staining, strongly suggest that the neuroinhibitory effects of recombinant MHC I were mediated through neuronal MHC I receptors.
The inhibition of neuronal outgrowth is a distinct biological activity from MHC I's previously described role in pruning, or stabilizing, synaptic connections (8
). We found that this neurologic activity required conformationally correct MHC I, evidenced by : 1) a mild heat treatment (that dissociated the complex) abolished recombinant MHC I's neuroactivity; and 2) a conformation-dependent anti-MHC I mAb removed the inhibitory neuro-activity. The presented peptide by itself had no neuroactivity on retina explants. Thus, retinal MHC I receptors are unlike the receptors of vomeronasal neurons, which can respond to small peptides (alone) that possess MHC I-binding motifs (37
Retina neurite outgrowth was inhibited by self-MHC I molecules, regardless of the peptide presented. The lack of specificity for the presented peptide may be a necessity; it is unlikely that a particular peptide would always be presented by different individuals' MHC I molecules, given that MHC I genes are highly polymorphic in outbred populations and each MHC I allele product binds different peptides. However, MHC I genes are differentially expressed temporally and spatially in the CNS (8
). Accordingly, quantitative changes in MHC I expression, rather than changes in the particular presented peptides, are likely to provide information for neurodevelopment. Indeed, in the innate immune system, NK cells can exquisitely distinguish between cells that differ only in their expression levels of MHC I.
We observed that retina neurite outgrowth was especially sensitive to inhibition by self-MHC I allele products. For example, on average, at 100 pM, H-2b molecules (but not H-2d or H-2k molecules) significantly inhibited neurite outgrowth from C57BL/6 retinas, and H-2d molecules (but not H-2b or H-2k molecules) significantly inhibited neurite outgrowth from BALB/c retinas. Interestingly, neurons from MHC I-deficient b2M−/− mice and Kb Db−/− mice were unresponsive to recombinant MHC I. These observations suggest that endogenous MHC I expression is required to sensitize retina neurons to self-MHC I.
How do neurons recognize MHC I given that MHC I is extremely polymorphic? Neurons could use a promiscuous MHC I receptor that is nonspecific for MHC I allele products. The PirB receptor in the visual cortex may represent such an indiscriminate neuronal MHC I receptor (10
). However, PirB may not be the RGC MHC I receptor, because PirB-deficient mice have normal retinogeniculate connections (10
). Alternately, neurons could express many MHC I receptors from a gene family, each with various MHC I allele specificities, such that at least one receptor can always recognize self-MHC I. Another possibility is that neurons undergo an “educational” process so that they express the correct MHC I receptors (out of a family of possible receptors), as occurs with Ly49 receptors on murine NK cells. Ly49 receptors are encoded by a large gene family encoding ≥23 different Ly49 receptors, each of which preferentially interacts with different MHC I types [e.g., Ly49A recognizes Db
, and Dk
; Ly49I recognizes Dd
; and Ly49D recognizes Dd
, and Dsp
)]; however, they have little or no specificity for the presented peptide (40
). It is believed that individual developing NK cells are “educated” to recognize self-MHC I by sequentially and cumulatively expressing different members of the Ly49 gene family until the cell expresses at least one receptor that interacts with self-MHC I (14
). In addition, it is believed that NK cells must interact with MHC I to be “licensed” to develop functional competence (42
). Notably, Ly49 is expressed by mouse cortical neurons (44
), but Ly49 is not expressed in humans. Hence, it is an open question whether mouse and human retinal neurons use an already known MHC I receptor(s), one or more of the many MHC I receptor-related genes whose function are not yet understood (45
), or a completely novel gene(s).
For endogenous MHC I to play a role in coordinating RGC recognition of MHC I with the inherited MHC I haplotype, MHC I and MHC I receptors must be expressed very early in retina development. Because the molecular identity of the retina MHC I receptor(s) is unknown and may be a novel receptor, we used an MHC I tetramer as a pan-specific probe for classical MHC I receptors in retina tissue sections. This MHC I tetramer staining, along with in situ hybridization and immunohistochemistry to localize MHC I, showed that MHC I receptors and MHC I are expressed in the same regions, and likely on many of the same cells, very early in retina development. The tetramer staining was particularly dense on retinal precursor cells and developing RGCs. In the middle retina region wherein several neuronal types reside, we could discern individual tetramer-stained cells, all of which also stained for anti-islet, indicating that these cells were developing RGCs migrating from the precursor layer to the GCL and not some other neuronal or immune cell type. The expression of MHC I and MHC I receptors in E12 retinas suggests that MHC I–MHC I receptor interactions may play a role during the early stages of retinal development. These interactions occur well before the RGC projections reach the thalamus and may prepare the developing RGCs to recognize MHC I on their target neurons. In addition to sensitizing RGCs to self-MHC I, MHC I and MHC I receptors in the developing retina may be involved in directing axon projections toward the optic nerve and in intraretina remodeling.
Our data suggest that inappropriate expression of MHC I on neurons could have deleterious consequences during neurodevelopment. It is believed that some neurodevelopmental diseases could arise from subtle abnormalities in axon path finding, axon number, synapse formation, and elimination. Many of the genes associated with human autism encode proteins involved in synaptic development (reviewed in Refs. 9
). Viral infection during pregnancy increases the risk for autism in humans (reviewed in Ref. 47
). Moreover, the infection of pregnant mice with virus or treatment with other immune stimuli cause the offspring to have autism-like behaviors (48
). These behavioral aberrations are believed to be due to maternal immune responses (e.g., inflammatory cytokines) that affected fetal neurodevelopment. Inflammatory cytokines, such as IFN-γ and TNF-α, induce neuronal MHC I expression (1
). Recently, the MHC region has been genetically implicated as a risk factor for schizophrenia (51
). In other studies, we observed that a modest increase in neuronal MHC I expression (30–60% higher levels) in transgenic mice could alter hippocampal electrophysiology, the levels of synaptic markers in their hippocampus, and compensatory neuronal sprouting responses (Z.-P. Wu, manuscript in preparation). Thus, modest changes in neuronal MHC I expression can have neurobiological consequences in vivo.
MHC I has bifunctional activities in the immune system: it can help to activate or inhibit immune responses, depending on the context. Likewise, many molecules involved in axon guidance are bifunctional, providing attractive or repulsive signals, depending on cellular conditions. Accordingly, in a different context, MHC I may promote neuronal outgrowth.
We showed that MHC I could inhibit neurite outgrowth, which may explain, in part, why MHC I expression is tightly regulated in the CNS. We have also begun to decipher the principles governing neuronal recognition of MHC I. Our results suggest that inappropriate neuronal MHC I expression could be deleterious during neurodevelopment. A further understanding of the roles that MHC I and MHC I receptors can play in the nervous system may lead to new classes of treatments to promote recovery in some neuropathological conditions.