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Abstract: PB2040

Type: Publication Only

Background
Specific microbial antigens have been implicated in the development and maintenance of several types of marginal zone lymphoma suggesting that an abnormal immune response is essential for driving B-cell proliferation. Reasoning that T-cell stimulation, especially from CD4+ T-cells, might make important contributions to promoting B-cell proliferation we carried out a detailed analysis of infiltrating T-cells in 8 cases of nodal marginal zone lymphoma. 

Aims
The aim of the study was to undertake the first detailed study of T-cell subsets in marginal zone lymphoma in order to determine the relationship of individual subsets to proliferating lymphoma B-cells.

Methods
We carried out multiplex immunohistochemistry and validated the results to show that, for the combinations of antibodies employed, there was firstly no reduction in intensity after several rounds of staining and destaining and secondly that there was no significant carry over from one round to the next. The stained slides were scanned and, utilising a custom macro written for ImageJ software, we enumerated CD4+ T-cell subsets (TH1- CD4⁺TBET⁺, Treg - CD4⁺FoxP3⁺, follicular helper (Tfh) - CD4⁺PD1⁺ and follicular regulatory (Tfr) - CD4⁺FoxP3⁺PD1⁺). 

Results
In all cases CD4⁺ T-cells constituted a major portion of infiltrating T-cells, mean 39.8% (range 13.5 to 70.3%). There were, however, large differences in the CD4⁺ T-cell subset composition; Tfh cells varied from 2.5 to 36% of all CD4⁺ T-cells whilst Tregs accounted for 2.7 to 24.7%. We also compared architecture of T-cell infiltration across cases and found that T-cells were not homogenously distributed and that CD4⁺PD1⁺ cell clusters could show some association or no association with CD4⁺FoxP3⁺ clusters and, in one case, repulsion from CD4⁺FoxP3⁺ clusters. In order to quantitate the associations we carried out Pearson correlations. For comparison normal tonsil showed a Pearson correlation of -0.4 i.e. no overalp, between CD4⁺PD1⁺ and CD4⁺FoxP3⁺ cells whereas there were varying degrees of association (range 0.3 to 0.8) for the lymphoma samples. By contrast proliferating Ki67⁺ lymphoma B-cells associated with CD4⁺PD1⁺ cell clusters (Pearson 0.1 to 0.6) whatever the relation to CD4⁺PD1⁺ cells to CD4⁺FoxP3⁺ cells. To confirm this result we used an alternative method to analyse clustering  (the Morisita index). This produced similar results with normal tonsil having a Morisita index of 0.2 for CD4⁺PD1⁺ and CD4⁺FoxP3⁺ whereas lymphoma samples showed higher degrees of association (range 0.3 to 0.9). The Morisita index also confirmed association between proliferating B-cells and CD4⁺PD1⁺ cells; normal tonsil 0.8 and lymphoma (0.4 to 0.8).

Conclusion
Collectively our data suggests an unsuspected association between CD4⁺PD1⁺ T-cells and proliferating lymphoma B-cells in marginal zone lymphoma.

Session topic: E-poster

Keyword(s): Marginal zone