Evaluation of immune infiltration in a population-based cohort of diffuse large B-cell lymphoma

Abstract

Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin lymphoma, characterized by an aggressive progression. Emerging evidence suggests that the tumor immune microenvironment (TIME), particularly tumor-infiltrating T cells, plays a key role in disease progression and prognosis for patients that are treated with modern combined immunotherapeutic regiments. Thus, the development of companion diagnostic strategies and workflow that allow systematic evaluation of the composition of the TIME in a high throughput manner is essential to develop.
This study evaluated the TIME in a population-based cohort of 650 DLBCL patients using a 6-plex multiplex immunofluorescence (mIF) panel targeting CD20, CD3, CD8, FoxP3, PD1, and DAPI. Due to technical issues, FoxP3 was excluded from the final analysis. Following optimization of antigen retrieval and amplification protocols, tissue microarrays (TMAs) were imaged and analyzed using InstanSeg and QuPath for segmentation and machine learning-based classification.
After quality control, 473 patient samples were included in the final analysis. High infiltration level of CD3⁺CD8⁺ and CD3⁺CD8⁻ T cells was associated with significantly improved 5-year overall survival (OS), confirmed by Kaplan-Meier and Cox regression analyses adjusted for age, stage, and International Prognostic Index. A subset of 55 patients with reliable PD1 staining showed that high levels of PD1⁺ T cells, both CD8⁺ and CD8⁻, were also associated with better prognosis.
InstanSeg outperformed Cellpose in both segmentation quality and processing speed, validating its utility for large-scale mIF image analysis. The study demonstrates that T cell infiltration, particularly CD8⁺ subsets, is an independent favorable prognostic marker in DLBCL. These findings underscore the importance of the TIME and suggest that image-based immune profiling may aid in patient risk stratification and treatment planning.

Popular Abstract

T cells in lymphoma tumors may help predict patient survival
In our study of over 450 lymphoma patients, we found that higher levels of certain immune cells inside tumors are linked to better survival. This discovery could help tailor treatment more precisely in the future.
Diffuse large B-cell lymphoma (DLBCL) is an aggressive blood cancer and the most common form of non-Hodgkin lymphoma, affecting 150 000 people each year. Despite advances in treatment, many patients relapse, and predicting outcomes remains a challenge. While current risk assessments mainly consider clinical factors like age and tumor stage, they often overlook the biology of the tumor itself—especially the role of the immune system.
Our thesis project focused on understanding the tumor immune microenvironment (TIME), the collection of immune cells that surround and infiltrate the tumor tissue. Using advanced imaging and statistical analysis, we studied tumor samples from 473 patients diagnosed with DLBCL in southern Sweden. Specifically, we looked at the presence of T cells, a type of immune cell known to fight infections and cancer.
There are several types of T cells in the immune system, which all play different roles. Cytotoxic T cells are the fighters of the immune system and have the capability to recognize and kill tumor cells. Helper T cells, another subtype, have a more coordinating role and activate other immune cells that can have a tumor suppressive role. However, depending on the specific microenvironment and signal molecules, T cells could also be used by the tumor to promote tumor progression.
We used a powerful technique called multiplex immunofluorescence to stain tissue samples with six different markers connected to one color each. This allowed us to detect multiple cell types in each tumor, visible in different colors.
After optimizing the laboratory process to stain the tissue, and after excluding poor-quality samples, we examined how many T cells were present in each tumor and whether this correlated with patient survival. We found that patients with high levels of cytotoxic and helper T cells lived longer than those with low levels of these cells. Importantly, this association held even after accounting for age, tumor stage, and other clinical factors. This means that T cell infiltration may help determine how long a patient will survive.
In a smaller subset of patients, we also analyzed a protein called PD1, which is often found on T cells that have lost their functions, such as killing of tumor cells. Surprisingly, we found that high levels of PD1⁺ T cells were also linked to better outcomes for the patients. This may indicate that these T cells were initially active before becoming exhausted and that these cells still play a role in fighting the tumor, despite expressing an inhibitory marker.
Our findings highlight that the immune system plays a crucial role in DLBCL and that digital analysis of tumor images can provide valuable insights into patient prognosis. In the future, this approach could help doctors personalize treatment by identifying patients more likely to benefit from immunotherapy or more drastic treatment.

• author: Wikström, Filippa and Hammer, Ida
• supervisor: Sara Ek; Angelica Johansson
• organization: Department of Immunotechnology
• course: KIMM05 20251
• year: 2025
• type: H2 - Master's Degree (Two Years)
• subject: Technology and Engineering; Biology and Life Sciences
• keywords: Diffuse Large B cell Lymphoma, DLBCL, immunotechnology, T cell, T cell infiltration, lymphoma, non-Hodgkin lymphoma, PD1, FoxP3, CD20, CD8, CD3, multiplex immunofluorescence, image analysis, tumor immune microenvironment, tumor microenvironment, TME, cytotoxic T cell, exhausted T cell
• language: English
• id: 9204014
• date added to LUP: 2025-06-24 13:38:01
• date last changed: 2025-06-24 13:38:01