The go-to metaphor for cancer drugs known as immune checkpoint inhibitors is a brake-release lever — a means of setting loose an immune system attack on tumor cells. Helpful as it is, the analogy glosses over a highly complex mechanism involving multiple types of immune cells, cell receptors, signals, and responses — so intricate that scientists have yet to dissect all of it.
In a new study, investigators at Dana-Farber unravel some key aspects of the immune system’s response to checkpoint inhibitors in the treatment of classical Hodgkin lymphoma. The findings, published in Nature Medicine, may provide a springboard to improving immunotherapies in Hodgkin lymphoma and other cancers as well.
“Immune checkpoint agents known as PD-1 inhibitors have revolutionized the treatment of classical Hodgkin lymphoma, with higher response rates than for any other form of cancer,” says Margaret Shipp, MD, the co-senior author of the study. “Despite this, it is not yet clear how these inhibitors work: which specific sets of immune system cells are unleashed to fight cancer and what stage of the immune response is most enhanced by them.”
In the new study, led by co-first authors Zumla Cader, MBChB, PhD, and Xihao Hu, PhD, and conducted by the Shipp and S. Liu groups, researchers set out to answer those questions.
A revived assault
PD-1 inhibitors work by making certain types of immune cells, particularly T cells, less passive in the face of cancer. As Dana-Farber’s Gordon Freeman, PhD, and Harvard Medical School’s Arlene Sharpe, MD, PhD, showed in research beginning in the 1980s, the PD-1 protein on T cells turns the cells into idle warriors: when PD-1 encounters its ligands, PD-L1 and/or PD-L2, on a tumor cell, this thwarts the T cell’s normal impulse to attack the tumor cell. Drugs that block PD-1 (or its counterpart proteins, PD-L1 and PD-L2, on tumor cells) can reawaken their fighting spirit.
But does PD-1 inhibition act equally on all T cells? Previous research suggested not.
There are two major classes of T cells: CD8+ T cells, also known as cytotoxic T cells, which are trained assassins of tumor cells; and CD4+ T cells, including T-helper, T regulatory, and additional T-cytotoxic subsets, which orchestrate a multi-layered attack on tumor cells. Hodgkin lymphoma has evolved a strategy to duck an attack by one of these classes. The surface of Hodgkin cells is largely barren of MHC Class I molecules, which display proteins called antigens to the immune system. Antigens signal the immune system to mount an attack on the antigen-presenting cell. CD8+ cells concern themselves with cells expressing MHC Class I molecules. Cells that don’t have them — including Hodgkin lymphoma cells — can fly under the radar.
“Research shows that in solid tumors, PD-1 blockade works by increasing the activity of CD8+ cells,” Shipp notes. “That’s unlikely to be the case in Hodgkin lymphoma, in which the tumor cells largely lack MHC Class I molecules.”
Other evidence supports the idea that CD8+ T cells play only a minor role in the immune response to Hodgkin. One study focused on MHC Class II molecules, which occupy the surface of Hodgkin lymphoma cells and present antigens exclusively to CD4+ T cells. The Shipp group previously evaluated tumor tissue from many patients treated with PD-1 inhibitors and found that high levels of MHC Class II molecules correlated with the best responses to the drug. When the investigators analyzed Hodgkin lymphoma tissue samples for a separate study, they found large numbers of CD4+ T cells but only a smattering of CD8+s.
“All this indicated a role for something other than classical CD8+ T cells — possibly for CD4+ cells — in the effectiveness of PD-1 blockade in Hodgkin lymphoma,” Shipp remarks.
Assessing the response
To determine if CD4+ T cells indeed lead the charge against Hodgkin lymphoma cells, Shipp and her colleagues drew on a cache of blood samples — 56 from patients with relapsed or recalcitrant forms of the disease who received a PD-1 inhibitor in a large clinical trial; some from patients diagnosed with, but not yet treated for, Hodgkin lymphoma; and some from healthy donors. The samples afforded a time-lapse look at how the immune system responds to the disease and to treatment with PD-1 blockers.
They focused on the T-cell receptor, a structure on the T-cell surface that probes antigens held by MHC molecules. The T-cell receptor consists of multiple subsegments arranged to conform to the shape of a specific antigen. These subsegments can be assembled in countless combinations, allowing receptors to be made for virtually any antigen lodged in an MHC molecule. A T cell may have as many as 20,000 receptors sticking up from its membrane, but they are all of a single type — all corresponding to a particular antigen.
For the Shipp and Liu teams, the T-cell receptor provided a lens into how well the immune system springs into action in response to PD-1 blockade. Specifically, they were interested in T-cell receptor diversity, the number of unique receptors within a given blood sample. Greater diversity equals a more nimble, versatile response, much as a socket wrench set with dozens of attachments can handle more jobs than a smaller set.
As Shipp puts it, “T-cell receptor diversity reflects the ability of T cells to potentially recognize multiple antigens and mount immune responses.”
To gauge T-cell receptor diversity, researchers analyzed the structural make-up of T-cell receptors in each blood sample, a process known as “receptor sequencing.” They sequenced the receptors on all the T cells within each sample and separately evaluated those on CD4+ and CD8+ T cells.
The investigators found that patients with Hodgkin lymphoma had significantly less T-cell diversity than healthy donors did, suggesting that a reduced T cell response plays an important role in the development of the disease. The decline was particularly pronounced in CD4+ T cells.
Other findings pointed to a connection between T-cell receptor diversity and the effectiveness of PD-1 blockade. “Patients who achieved the most favorable responses to blockade — whose cancer became undetectable after treatment — had higher initial diversity than those with only partial responses. And patients who had no response to PD-1 blockade had lowest initial diversity,” Shipp comments.
Investigators also looked at changes in CD4+ and CD8+ T cell diversity during treatment with PD-1 inhibitors. “We found that treatment was associated with a highly significant increase in CD4+ cell diversity but no increase at all in the diversity of CD8+ cells,” Shipp observes. “This is strong evidence that CD4+ cells are key actors in the immune response to PD-1 blockade.”
Generalists and specialists
Next, investigators explored whether the effectiveness of PD-1 blockade is linked to the stage of the immune response to Hodgkin lymphoma: Does it primarily spur less-mature T cells that are capable of taking on a range of cellular foes, or does it spark more specialized T cells that have committed to a particular enemy? This involved conducting what’s known as a “clonal expansion analysis.” Researchers identified the T-cell receptors in blood samples from patients who had yet to undergo treatment and divided them into two categories: receptors for which there was just one copy (dubbed “singletons”), and those for which there were multiple copies (“non-singletons”). They performed the same analysis on samples taken after patients had been treated, and determined which group showed the biggest increase in numbers and activity.
“Earlier research suggested that PD-1 blockade reactivated or expanded the more differentiated non-singletons,” Shipp states. “We found the opposite: there was a highly significant association between the expansion of singleton clones and a favorable response to PD-1 blockade. This suggests that PD-1 blockade is likely working at an earlier timepoint than had been appreciated. And it aligns with recent findings that suggest the most specialized, differentiated T cells can’t be reinvigorated or reactivated.”
The research turned up one more surprise: untreated patients had a substantial decrease in circulating natural killer (NK) cells — immune system cells that are among the body’s first responders to disease and infection. Intriguingly, patients with the highest numbers of an NK cell subtype had some of the best responses to PD-1 blockade.
“These results allow us to consider ways of complementing current PD-1-targeting therapy with other drugs that may enhance its effectiveness,” Shipp says. “In many ways, Hodgkin lymphoma serves as a model of how tumor cells evade an immune system attack, so our findings may be put to use in enhancing immunotherapy for other cancers as well.”