Page 144 - Haematologica - Vol. 105 n. 6 - June 2020

W. Townsend et al.
close spatial association of Ki67pos B cells and TFH is recapit- ulated in FL, as 41% of Ki67pos FL B cells were in direct con- tact with TFH and were significantly more likely to be in direct contact with TFH than non-proliferating cells. The observed close spatial correlation between the two cell types is thus not due to chance and suggests that TFH are involved in functionally important interactions with the tumor. This corroborates and advances findings from previ- ous in vitro experiments which showed that FL TFH provide signals for B-cell survival.25,27
We also found a correlation between the numbers of TFH and Ki67pos B cells in both normal GC and neoplastic folli- cles and that, in FL, the number of TFH increase with histo- logical grade. The relationship between number of TFH and rate of B-cell proliferation observed in our study has not been reported previously in FL but is consistent with previ- ous data showing that the regulation of GC size and B-cell number is critically dependent on the number of TFH.17,19,23 This adds to the evidence that TFH are central to the patho- genesis of FL, just as they are essential in the normal GC reaction. Furthermore, the degree of co-localization (the proportion of Ki67pos B cells in contact with one or more TFH) remained constant as histological grade increased, with no significant change in the proportion of Ki67pos cells in con- tact with TFH in grade IIIa or IIIb disease compared to grade I-II disease, suggesting that interaction with TFH remains important regardless of histological grade.
Our studies also underline the crucial importance of using a multi-parameter approach to define and quantify the com- plex T-cell subsets present in the FL microenvironment. No single antigen or transcription factor specifically identifies TFH and this is the first study reporting the presence of TFH in FL in situ using techniques that overcome the limitations of traditional IHC. By using co-staining for ICOS and BCL6 we were able to show that only half of the PD1 expressing cells neoplastic follicles are TFH. Single parameter analysis of PD1 would therefore lead to significant overestimate of TFH num- bers perhaps explaining, at least in part, why previous IHC studies have yielded divergent results with regards to the impact of different T-cell infiltrates on prognosis.10,13-16
Previous in vitro studies have shown that peripheral blood T cells in FL are dysfunctional and form impaired synapses with B cells.40,42 In the present study, however, we found features that suggest normal synapse formation between Ki67pos tumor cells and TFH within the LN.43 This divergence from previous research may be because we examined the interactions between TFH and Ki67pos cells in situ in human tissue rather than in an ex vivo system using peripheral blood derived cells. It also remains possible that there are other subsets of non TFH cells in the FL microenvironment that are dysfunctional and have an impaired ability to form immunological synapses.
In addition to promoting GC B-cell proliferation, interac- tion with TFH cells also induces AID expression which induces somatic hypermutation and class switch recombi- nation. Off-target action of AID has previously been pro- posed to lead to the accumulation of mutations required for germinal center-derived lymphomas to develop or progress and has been associated with transformation of FL.44,45 The close spatial association between TFH and AIDposKi67pos FL B cells observed in the present study is compatible with this theory.
Finally, next generation sequencing analysis of the TCR repertoire of follicular and interfollicular areas of FL LN showed that the neoplastic follicles are significantly more clonal and dominated by high frequency clones compared to the interfollicular regions. As expected from their divergent phenotype, very little repertoire overlap between the two compartments was present. PCR-based analyses of TCR repertoire on small samples are known to suffer from a number of potential limitations including sampling effects and errors introduced during the ampli- fication process, which may lead to apparent skewing.46 Whilst we cannot completely exclude these possibilities, we minimized the risk by direct, intra-patient compari- son in the same assay run, and, of note, our findings were consistent in all five cases studied. Another possibility is that the demonstrated differences in TCR repertoire relate to the greater number of T cells found in the inter- follicular regions compared to the follicles. Whilst T-cell numbers undoubtedly do differ between these two areas, significant difference in the clonality index, which takes into account the number of unique clones present, were observed (Figure 6A). Furthermore, the repertoire of the intrafollicular area was strikingly dominated by high fre- quency clones; for example, the top 50 clones accounted for a mean of 19% (CI: 17-21%) of all clones present, compared to 9.7%(CI: 6.1-13.4%) in the interfollicular areas (P=0.0002).
Taken together, these findings suggest that the interac- tions between B cells and activated TFH that induce B-cell proliferation and differentiation and lead to the generation of high affinity antibody in normal GC may be recapitulat- ed within the follicles of FL.19,41 Since TFH may be involved in processes fundamental to disease progression, such as clon- al expansion and genomic evolution of the tumor, our results suggest that they would be an attractive target for novel therapies. This is especially relevant in the era of drugs that target antigen receptor signaling such as PI3- kinase inhibitors, which affect both B- and T-cell receptor pathways. Our results are also relevant to understanding the mechanism of action of drugs that target PD-1 expressing cells, which have been shown to be effec- tive in FL and other lymphomas.29-31 It is clear that whilst some of the PD-1 expressing cells in the FL LN are indeed TFH, many are not and these may represent exhausted effec- tor cells. Blockade of PD-1 in the latter case may unmask antitumor immunity and lead to disease regression. The impact of interrupting PD-1 function in TFH is, however, less clear as the role of the PD-1 axis in TFH function is not fully established. These findings add another level of complexity to our understanding of the FL tumor microenvironment and underline the necessity of using multi-dimensional methods in future studies.
Funding
This research was funded by grants from the British Society of Haematology in partnership with the Roger Counter Foundation, and Bloodwise.
Acknowledgments
We are grateful for the technical assistance and advice of Adaptive Biotechnologies and of Jon Harris and Jan Soetaert from the Nikon Imaging Centre at Kings’ College London.
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