A team including University of Wyoming anthropologists works at the site of a circular plaza that was built around 4,750 years ago in the Cajamarca Basin of northern Peru.

Two University of Wyoming anthropology professors have discovered one of the earliest circular plazas in Andean South America, showcasing monumental megalithic architecture, which refers to construction that uses large stones placed upright with no mortar.

Located at the Callacpuma archaeological site in the Cajamarca Basin of northern Peru, the plaza is built with large, vertically placed megalithic stones—a construction method previously unseen in the Andes. Associate Professor Jason Toohey, project lead, and Professor Melissa Murphy have been researching this topic since the project’s inception in 2015. Excavations took place in the plaza starting in 2018.

Their paper, which reports new data on this earliest known megalithic circular plaza in the northern Andes, is titled “A Monumental Stone Plaza at 4750 BP in the Cajamarca Valley of Peru” and has been published in Science Advances.

Radiocarbon dating places its initial construction around 4,750 years ago during the Late Preceramic Period, making it one of the earliest instances of such architecture in the Americas.

To better understand this timeline, the team carefully excavated within the plaza, uncovering artifacts related to life in the past and collecting charcoal samples for dating. All material remains then were cleaned, processed and analyzed in the laboratory.

“This structure was built approximately 100 years before the Great Pyramids of Egypt and around the same time as Stonehenge,” Toohey says.

These dates signify that the circular plaza at Callacpuma is the earliest known example of monumental and megalithic architecture in the Cajamarca Valley—and one of the earliest examples in ancient Peru.

“It was probably a gathering place and ceremonial location for some of the earliest people living in this part of the Cajamarca Valley,” Toohey adds. “These people were living a primarily hunting-and-gathering lifestyle and probably had only recently begun growing crops and domesticating animals.”

The plaza is formed by two concentric walls and measures about 60 feet in diameter.

The project is led by Toohey and Patricia Chirinos Ogata from the University of California-Santa Barbara. The team also includes Murphy, as well as undergraduate and graduate students from Peru and the U.S.

Toohey is an anthropological archaeologist who is dedicated to taking a holistic and multidisciplinary approach to the field. He has conducted fieldwork in the Peruvian Andes since 2003. The department head for anthropology at UW, Murphy is a biological anthropologist specializing in bioarchaeology and committed to multidisciplinary approaches within anthropology.

“As part of our community outreach, we collaborate and work with the residents of the towns on and adjacent to the site of Callacpuma about our findings and their importance,” Toohey says. “We highlight the importance of cultural heritage, and working together, we can continue the scientific investigations and help to preserve the site.”

Cervical cancer is among the most common malignancies affecting women worldwide. In 2020 alone, approximately 600,000 women were diagnosed with this disease, and over 314,000 died from it. In 99% of the cases, cervical cancer cells harbor human papillomavirus (HPV), and thus, HPV vaccines are an effective way to mitigate the risk of developing this disease. Unfortunately, such preventive measures are useless against established cancers, which are generally incurable once they become metastatic or relapsing.

Fortunately, scientists have made substantial progress in developing a promising therapeutic strategy for cervical cancer: rejuvenated cytotoxic T lymphocytes (rejTs). These lymphocytes can be engineered to target HPV-specific antigens that are expressed predominantly in cervical cancer cells, constituting a type of targeted immunotherapy. Ideally, rejTs would be produced from induced pluripotent stem cells (iPSCs) gathered from the patient themselves. However, this process is not clinically feasible in terms of both time and cost.

Against this backdrop, a research team including Chief Professor Miki Ando, graduate student Yoshiki Furukawa, and Assistant Professor Midori Ishii from Juntendo University School of Medicine, Japan, has recently achieved a breakthrough by developing robust iPSC-derived rejTs for cervical cancer treatment. Their work was published in Cell Reports Medicine.

The team sought to address one of the key roadblocks for allogeneic iPSC-derived rejTs, which refers to rejTs produced from ‘standardized’ iPSCs rather than derived from the patient’s cells. Prof. Ando explains, “In immunocompetent cervical cancer patients, the dominant problem is the rejection of foreign T lymphocytes by the recipient’s CD8⁺ T lymphocytes or natural killer (NK) cells.” The patient’s immune system tends to attack the therapeutic HPV-specific rejTs before they can neutralize cancer cells.

The research team used CRISPR-Cas9 two-step “scarless” gene editing on iPSCs derived from an HPV-specific cytotoxic T lymphocyte clone to overcome this issue. The first modification was the deletion of all HLA class I antigens from the cells. The role of these surface proteins is to present peptides to CD8⁺ T lymphocytes, which promptly eliminate any cells displaying foreign or anomalous peptides. After the modification, the cells are essentially able to evade detection by CD8⁺ T lymphocytes.

The second modification involved introducing the limited expression of two specific HLA antigens, namely HLA-A24 and HLA-E. This enabled the engineered cells to evade attacks from NK cells, which specifically target cells lacking these surface antigens. By selecting HLA-A24, the engineered cells are naturally compatible with a significant portion of South American, Eastern Asian, North American, and European populations.

After implementing these modifications using CRISPR-Cas9, the researchers induced the differentiation of the iPSCs into T lymphocytes and tested their ability to fight cervical cancer cells while evading allogeneic immune responses both in vitro and in vivo. These experiments yielded very promising results, as the generated rejTs did not trigger attacks from either CD8⁺ T cells or NK cells while simultaneously achieving strong cytotoxicity against tumor cells.

Compared to the control group, mice engrafted with cervical cancer cells and injected with the gene-edited rejTs survived longer and exhibited significantly reduced tumor sizes and proliferation index. To gain further insights into the enhanced therapeutic effects of the rejTs, the researchers conducted single-cell RNA sequencing analyses. These analyses revealed that the population of rejTs was highly enriched with tissue-resident memory T cells, which establish residence in the mucosa of the cervix and provide stronger protection.

Prof. Ando remarks, “We found that the enhanced cytotoxicity against cervical cancer occurred through TGFβ signaling and increased CD103 expression.”

Taken together, the results of this study showcase a powerful method to make iPSC-derived rejTs a viable option to treat one of the most common forms of cancer. Prof Ando concludes, “The HLA-engineered HPV-rejTs obtained using our method provide a sustainable and promising approach toward successful ‘off-the-shelf’ T cell therapy, which could help in overcoming cervical cancer. We are planning on conducting an investigator-initiated clinical trial in 2024.”