SLAMF7 and IL-6R distinguish cytotoxic from non-cytotoxic T cells . 63

In order to identify further markers that can distinguish cytotoxic from helper-type T-cell subsets we screened for genes that were differentially expressed in the cytotoxic T-cell populations (Tc1, Tc17+1) compared to all other non-cytotoxic Tc and Th subsets. We could identify 28 genes that were more weakly and 22 genes that were more strongly expressed among the cytotoxic T cell subsets (Figure 32A). Strikingly, the two cell surface molecules SLAMF7 and IL-6R displayed a converse expression pattern among all lymphocytes (Figure 32B). In concordance, all CD4⁺ as well as CD8⁺ Tc2, Tc17 and Tc22 helper T-cell subsets but not the cytotoxic Tc1 and Tc17+1 cell subsets expressed high levels of IL-6R (Figure 32C). Opposing expression patterns were observed for SLAMF7 that was only expressed by the cytotoxic Tc1 and Tc1+17 CD8⁺ memory T-cell subsets and all Granzyme B and/or Perforin expressing CD8⁺ T cells (Figure 32 D). Naive

CD8⁺ and CD4⁺ T cells lacked SLAMF7 but expressed IL-6R, whereas cytotoxic CD8⁺ effector T cells expressed the highest levels of SLAMF7 (Figure 32E).

SLAMF7 was co-expressed on all CD8⁺ T cells expressing Granzyme B, Perforin and/or IFN- after in vitro activation (Figure 32F). Moreover, SLAMF7 is detectable among Granzyme B⁺ Perforin⁺ cytotoxic CD4⁺ T cells (Figure 32F).

Since all further cytotoxic subsets among lymphocytes, the CD56 expressing NK, NKT and ILC1 cells were SLAMF7⁺IL-6R^- (Figure 32G), SLAMF7 represents a highly suitable marker to assess the cytotoxic potential of lymphocytes while IL-6R represents the non-cytotoxic fraction.

Figure 32: SLAMF7 and IL-6R expression distinguishes cytotoxic memory CD8⁺ and CD4⁺ T cells from non-cytotoxic cells.

(A) Heatmaps of normalized and scaled genes distinguishing cytotoxic (Tc1 and Tc17+1) and non-cytotoxic CD8⁺ and CD4⁺ T cell subsets (Tc2, Tc17, Tc22, Th1, Th2, Th17, Th17+1, Th22). (B) Representative dot plot of IL-6R and SLAMF7 expression among lymphocytes.

(C, D) Flow cytometric stainings indicating frequency and MFI of of IL-6R (C) and SLAMF7 (D) among CD45RA^-CD4⁺ and CD8⁺ T cell subsets. (E) MFI of of IL-6R and SLAMF7 of CD45RA⁺CCR7⁺ naïve CD4⁺ and CD8⁺ T cells and CD45RA⁺CCR7^- effector CD8⁺ T cells (F) Flow cytometric co-stainings of SLAMF7 with Granzyme B, Perforin and IFN- after 6hrs of polyclonal activation in the presence of BrefA, gated on CD8⁺ T cells or CD4⁺ T cells. (G) Representatitve dot plot of SLAMF7 and IL-6R coexpression with CD56 among lymphocytes.

3.5.5 CD40L⁺ memory CD8⁺ T cell subsets possess a unique TCR repertoire

Previous studies have suggested a relative plasticity in cytokine production and lineage transcription factor expression among differentiated CD4⁺ memory T-cell subsets (Kunicki et al., 2017; O’Shea and Paul, 2010). Becattini et al. furthermore demonstrated heterogeneous differentiation of pathogen-specific CD4⁺ T cells into distinct CD4⁺ helper T cell sister clones (Becattini et al., 2015). However our CD40L expression pattern suggests a preferential differentiation of CD40L⁺ naive CD8⁺ T cells into Tc2, Tc17 and Tc22 memory CD8⁺ T cells. To assess a potential corporate contribution of the different CD8⁺ T-cell subsets to specific immune responses, we performed TCRβ-chain deep sequencing of isolated Tc subsets.

The relative numbers of clonotypes were smaller in Tc17 and Tc22 cells compared to Tc1, Tc2 and Tc17+1 with a few clones dominating their repertoire (Figure 33A, B). While less than 0.6% of the clones are present in frequencies above 1%, they accounted for 60% (Tc17) and 90% (Tc22) of the reads (Figure 33C, D). The oligoclonal repertoire in Tc17 and Tc22 cells with a few dominating clones suggests a highly specialized response in contrast to the rather polyclonal Tc1, Tc17+1 and Tc2 cells.

Figure 33: Some clones are enriched among Tc17 and especially Tc22 cells.

Tc1, Tc2, Tc17, Tc17+1 and Tc22 were sorted from human PBMC and analyzed by TCR-sequencing. (A) Number of clones found in different subsets. Mean ± SEM. (B) Number of clonotypes and their frequency of reads of one representative donor. (C) Number of clonotypes present at frequencies >1% in the total clonotype pool. (D) Cumulative frequency of all clonotypes present at frequencies >1%.

Next, we asked whether the oligoclonality of Tc17 and Tc22 cells is caused by a limited selection of TCRs, which would suggest a detection of invariant MHCs (MHC-Ib). We therefore assessed the TCR-V usage of the different donors among naïve and the different Tc subsets (Figure 34). The V families varied among donors as well as among Tc17 and Tc22 indicating they detect diverse antigen:MHCs combinations. However, Tc17+1 cells displayed a strong enrichment for TCR-V chains of the V6 family and therefore might recognize antigens presented in a MHC-Ib dependent manner.

Figure 34: TCR-V familiy usage verifies TCR polyclonality among Tc17 and Tc22 T cells.

Pie charts of the percentage of the different V family contributions to the total TCR repertoire of naïve CD8⁺ T cells and memory Tc subsets.

The clone repertoire revealed that most clones overlap within the two cytotoxic subsets Tc1 and Tc17+1 and within the three non-cytotoxic subsets Tc2, Tc17 and Tc22 (Figure 35A). In total 5-10% of the clonotypes and around 50% of the

reads were shared by the cytotoxic and by the helper-type subsets, respectively (Figure 35B, C). Given the small overlap between those two groups in their shared clonotypes and shared reads, cytotoxic and helper-type CD8⁺ T cells detect distinct epitopes and were most likely involved in diverse antigenic responses. However, within such response heterogeneous differentiation or plasticity allows the differentiation of either Tc1 or Tc17+1 cells and accordingly either Tc2, Tc17 or Tc22 cells.

Figure 35: The clone repertoire overlaps among the cytotoxic and among the non-cytotoxic CD8⁺ memory T cell subsets.

(A) Venn Diagram of an example donor visualizes number clonotypes shared among different Tc subsets. (B) Summary of clonotype numbers shared among different Tc subsets from all donors. (C) Summary of reads shared among different Tc subsets from all donors.