Background/Purpose: CD6, expressed on T cells and 50% of NK cells, is emerging as a novel treatment target for both cancer and autoimmune diseases. We first showed that CD6 deficiency leads to significantly attenuated disease severity in murine models of multiple sclerosis, uveitis, and rheumatoid arthritis, in all of which autoreactive CD4+T cells are the primary drivers of pathogenesis, suggesting CD6 on CD4+T cells as a potential therapeutic target for these autoimmune diseases. Indeed, in CD6-humanized mice treated with UMCD6, an anti-human CD6 monoclonal antibody, striking reductions in clinical signs of disease, pathogenic Th1/Th17 responses, and inflammatory cell infiltration into the target organs were observed. UMCD6 works in a different way as a cancer immunotherapy agent, altering the gene expression pattern of cytotoxic CD8+T and NK cells to augment their cytolytic capabilities. Importantly, UMCD6 does not deplete T-cells but instead, leads to the rapid internalization of CD6. In assessing whether other lymphocyte surface structures co-internalize with CD6, we recently found that CD28 expression on the cell surface is robustly reduced in a sustained manner, especially on CD4+T cells.

Methods: Human PBMCs were isolated from healthy volunteers and cultured in RPMI media + 10% FBS with or without UMCD6 (10µg/ml). CD2, CD3, CD4, CD6, CD28, CD40L, and CTLA-4 expression was measured by Flow Cytometry using antibodies from BioLegend: CD2 (TS1/8), CD3 (UCHT1), CD4 (OKT4), CD6 (BL-CD6), CD28 (CD28.2), CD40L (24-31), and CTLA-4 (BNI3).

Results: UMCD6 rapidly depleted CD6 from the cell surface of T cells and NK cells. Cell surface expression of CD28 and CD2 was concurrently reduced (Figure 1A, B, C). CTLA-4 and CD3, but not CD4 were also reduced (1B). These changes persisted for at least 72 hours. The decrements in the various structures were most striking on T cells, somewhat less on NK-T cells, and not seen with NK cells (other than >90% reduction in expression of CD6). At baseline, very few CD4+ cells are CD28-negative, but many of CD4-negative (mostly CD8+) cells are CD28-negative (42%). This proportion increases to 58% by 24 hours following exposure to UMCD6. The change in CD28 expression on CD4+ cells is best appreciated as a decline in mean fluorescence intensity in the UMCD6 group compared to the IgG control. The reduction in CD28 MFI was more significant among CD4+ cells compared to CD4-negative cells (1C).

Conclusion: Considering the function of CD28 as a receptor for the second signal that leads to activation of CD4+T cells, with CD2 as a potential alternate second signal, alteration of CD28 and CD2 expression by UMCD6 is an attractive explanation for the effect of UMCD6 on autoimmune diseases and is potentially a significant step towards immune tolerance. Since many cytotoxic CD8+T cells are CD28-negative at baseline, further loss of surface CD28 on the CD8+ subset caused by CD6 internalization is not expected to compromise function of these cells. These results provide an explanation for the ability of UMCD6 to selectively inhibit CD4 cell function while concurrently stimulating cytotoxic lymphocytes to kill cancer cells, thereby offering the prospect of a new cancer immunotherapy that suppresses rather than triggers autoimmunity.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/reduction-in-cd4t-cell-expression-of-cd28-and-cd2-by-antibody-induced-internalization-of-cd6-a-potential-step-towards-immune-tolerance/