Anti-CTLA-4 immunotherapy creates new T cells while freeing them to fight

Nobel Laureate Jim Allison, Ph.D., invented immune checkpoint blockade immunotherapy by blocking the CTLA-4 protein on T cells, freeing those killer immune cells to attack cancer. Now Allison and colleagues have shown that thwarting CTLA-4 also liberates T cells to assume new identities, including one that’s vital to an effective response against tumors.

In a paper in the April edition of Immunity, the researchers show that CTLA-4 plays a role in peripheral CD4 T cell differentiation – a naïve cell’s transformation into a specific type of T cell – in addition to the protein’s established role as a “brake” suppressing T cell activation.

“Our findings tell us that we may be creating entirely new T cells with CTLA-4 inhibition, not just expanding existing cells,” says Allison, professor and chair of Immunology at MD Anderson and winner of the 2018 Nobel Prize in Physiology or Medicine.

They found that T helper cells – T cells that express the CD4 protein on their surface and perform a variety of functions depending on their differentiation from their initial, naïve state – are affected by CTLA-4.

CD4 T cell is essential to anti-tumor response

The team used mass cytometry to profile T cells in mice with CTLA-4 genetically deleted and compared them to those found in mice with normal CTLA-4. Three specific phenotypes of T cell existed only in the mice lacking CTLA-4. All three are positive for expression of inducible co-stimulator (ICOS) protein on the cell surface and one matches a cell type shown to be vital to effective anti-tumor immune response with Allison’s original checkpoint blockade therapy.

Previous studies initiated in 2006 by current study co-author Padmanee Sharma, M.D., Ph.D., professor of Genitourinary Medical Oncology and Immunology, identified ICOS-positive CD4 T cells in patients with bladder cancer who were treated with anti-CTLA-4 antibody. Subsequently, Sharma demonstrated in  pre-clinical studies in mice that the ICOS-positive cells were crucial for optimal anti-tumor activity.

ICOS-positive CD4 cells invaded tumors and increased the ratio of effector T cells, those involved in immune response, to regulatory T cells (Tregs), which suppress anti-tumor immune response, Sharma found. The ICOS-positive CD4 cells also wielded interferon gamma and tumor necrosis factor alpha, cytokines that fire up immune responses.

“This was considered a weird finding back then, because before that, ICOS had been found to be expressed mainly on regulatory T cells and follicular helper T cells,” Allison says.

Sharma discovered the ICOS-positive CD4 cells were found only in the tumor microenvironment and peripheral circulation but only after treatment with ipilimumab. She later established the signaling pathway used by ICOS to direct the cells to a specific immune-boosting type called Th1.

“The question was, where do these cells come from?” Allison says. The technology to explore the cells’ origin was not available until recently.

Using mass cytometry, the team profiled T cells that arise when CTLA-4 is absent and identified clusters of CD4 T cells unique to mice in which CTLA-4 was genetically deleted. 

To further confirm this finding and understand how these cells are generated, they then applied a different technique called archetypal analysis. They identified a shift in the boundary of CD4 T cell phenotypes, new types of T cells that can only develop without intact CTLA-4 signaling. These include three types of CD4 T cells:

• ICOS-positive TBET-positive CD4 effector T cells analogous to those identified by Sharma.
• ICOS-positive CD44-positive CD62L-positive CD4 regulatory T cells.
• ICOS+ BCL6+ GATA3+ CD4 effector follicular helper T cell (Tfh) vital to immune B cell function.

Allison said the researchers don’t know what function the new regulatory T cell or the Tfh cell archetypes perform, although neither Tregs nor Tfh play a positive role in the tumor microenvironment.

Super-producers of cytokine weapons

Having looked at the phenotypes of T cells, the team conducted cytokine profiling to see if the function of the new archetypal cells is affected by the absence of CTLA-4.  They found a major effect in both of the effector CD4+ T cell archetypes.

“CTLA-4 knockout increased both the frequency of cytokine-producing cells and the maximum level of cytokines produced per cell,” says first author Spencer Wei, Ph.D., a post-doctoral fellow in Allison’s lab and now a senior scientist at Spotlight Therapeutics.

Allison and Wei characterized the cells as reaching “super-physiological” level of cytokine production. “These cells have an extended phenotype that’s associated with extreme function,” Wei says.

The new archetypal Tregs showed an increase in cytokine-producing cells but no greater production of cytokines per cell.

Knocking out CTLA-4 had no effect on phenotypes of CD8-positive cytotoxic T cells. Knocking out another checkpoint, PD-1, had a slight effect on the mix of phenotypes of CD8-positive cells, but did not result in the appearance of any CD8 T cells that were not found in normal mice.

Having shown these effects on mice with CTLA-4 completely absent, the researchers then used antibody blockade to inhibit CTLA-4 in mice and found that treatment also enriched specific types of CD4+ effector T cells.

Overall, the team’s findings indicate that CTLA-4 regulates both T cell activation and T cell differentiation. These data indicate CTLA-4 activity, in addition to cytokine signaling and T cell receptor signal strength, regulates peripheral T cell differentiation, providing a new mechanistic insight for research to develop next-generation cancer immunotherapies. 

Sharma and Allison lead major immunology research efforts at MD Anderson. Allison is executive director and Sharma Scientific director of the immunotherapy platform and they are director and co-director of the Parker Institute for Cancer Immunotherapy.

Funding for this research was provided by MD Anderson’s National Cancer Institute Cancer Center Support Grant (P30CA16672), a grant from the Cancer Prevention & Research Institute of Texas, and MD Anderson’s Bioinformatics Shared Resource. Wei was an MD Anderson Odyssey Fellow.