## 4 Discussion
Analysis of gene expression levels for each marker showed few differences in expression levels for these macrophage markers between CON and LDP groups, despite the higher numbers of cells expressing each marker in the LDP samples compared to CON (Figure 5b). Macrophages can also undergo polarization into the classically activated pro-inflammatory phenotype, M1, or the alternative anti-inflammatory phenotype, M2 [39; 56]. They have a remarkable range of cellular functions that can be elicited during tissue injury and repair dependent upon macrophage polarization within the affected tissue [57]. M1-typemacrophages are classically activated and differentiation is stimulated by IFNy to secrete pro-inflammatory cytokines such as IL6, IL12, and TNF\(\alpha\). M2-type macrophages are alternatively activated macrophages, and their differentiation is stimulation by IL4 produced by Th2 T lymphocytes. Their roles are to secrete anti-inflammatory cytokines and factors such as Arginase 1, IL10, and aid in wound healing, tissue repair, and regeneration. The myeloid cluster was highly enriched for both M1 macrophages and M2 macrophages at 2 weeks post IVD injury (Figure 5c,d). While both M1 and M2 macrophage markers are present, there is a significantly higher expression for _Cd86_ (M1) and significantly lower expression for _Arg1_ (M2) genes, suggesting a shift towards M1, or pro-inflammatory state with LDP at 2 weeks post-injury. This shift away from the M2 phenotype is consistent with the observations in the human degenerate nucleus pulposus cells that exhibited declining M2 proportion with increasing degeneration severity. Single-cell transcriptomic analyses of the human IVD cells show also the increasing activation of immune recruitment pathways during degeneration, demonstrating the robust interactions between the immune system and the degenerating IVD. Our study here also identified the increased activation of B- and T-lymphocytes two weeks into the degenerative process. The majority of the top 25 commonly upregulated genes were in the myeloid and lymphoid clusters, and gene analysis led to the intriguing discovery that over 80% percent of those genes were highly enriched in lymphoid cell lineages. Notably, B cell-associate gene markers were the most represented group (40%: _Pika3p1_, _Rda2_, _Blink_, _Syk_, _Izf3_, _Mzb1_, _Cd19_, _Mybl1_) while the T cell-associated gene markers were slightly less prominent (25%: _Cufp2_, Lef1, _Mybbp1a_, _Prag1_, _Mybb_). Several gene markers were expressed in both T- and B- cells (35%: _Pptrcap_, _Sash3_, _Rag1_, _Top2a_, _Mybl2_, _Cd53_, _Laptm5_) (Figure 4d). B-lymphocyte specific markers included _Blink_, _Rac2_, and _Pik3 ap1_, which are involved in receptor signaling; _Syk_, _Cd72_, _Ikef_, and _Cd19_, which are involved in differentiation and proliferation; and _Mzb1_, _Mybl1_, which are involved in antibody production and secretion. Due to the large presence of lymphocyte genes, we checked for the presence of B and T lymphocyte specific markers within the lymphoid clusters to confirm these immune cell types were present. B cell co receptor and receptor, _Cd19_ and _Cd79b_, were present along with _Blink_, which plays a role in B cell maturation; _Cd38_, which is expressed on B cell progenitors and plasma cells [58; 59]; and _Ccx74_, which plays a role in B cell activation and trafficking (Figure 5e) [60]. T lymphocytes cell receptor, _Cd3g_, had increased gene expression levels and an increase in cell number. Interestingly, only a small number of cells expressed _Cd4_, helper T cells, or _Cd8b_, cytotoxic T cells, emerged as identifiable in the LDP samples (Figure 5f). Macrophages are capable of expressing cytokines that activate T-cells so that we examined T-cell specific markers for the incidence and genotype of T-cells. Notable T cell-specific markers were _Cufp2_, needed for T cell adhesion; _Lef1_, required for IL17A expressing T cell maturation; and _Myb_/_Mybbp1a_, which are involved in T cell differentiation.