Long-term exposure to RTX impacts on NK cell function and phenotype
Initial studies evaluated the impact of RTX on ADCC as well as the number and phenotype of NK cells in longer term cultures of PBMCs and Raji cells. The elimination of target B cells by RTX over time suggested ongoing ADCC (Fig. 1a). NK cells in these cultures persisted and proliferated as demonstrated by the maintenance in NK cell number (Fig. 1b) and CFSE dilution (Fig. 1c). After 5–7 days, NK cells within PBMCs cultured with Raji cells and RTX shifted from CD56dim to CD56bright (Fig. 1d, e). This change was not seen when TRA was added instead of RTX. OBZ, an anti-CD20 mAb recognizing a different-oriented epitope from RTX, showed changes consistent with those seen with RTX (Fig. S1a, b). Change in NK cell phenotype in vivo was determined using peripheral blood samples from patients receiving weekly single agent RTX (Fig. 1f). The fraction of NK cells with the CD56bright phenotype increased following RTX in the patient with circulating malignant cells but not in the patients without circulating malignant cells (Fig. 1g, Fig. S1c). Further studies were done to assess whether the shift in CD56dim and CD56bright NK cells derived from the differential expansion of the two subsets, or a shift of CD56dim to CD56bright cells. Sorted CD56dim and CD56bright NK cells were labeled with CFSE and CellTracker Red, respectively. Labeled CD56dim and CD56bright NK cells were added back to autologous PBMC and cultured with Raji cells and RTX (Fig. 2a). After 7 days, CFSE-labeled CD56dim NK cells proliferated and displayed a CD56bright immunophenotype (Fig. 2b), while CellTracker Red-labeled CD56bright cells did not expand (Fig. 2c). These studies demonstrate CD56dim NK cells within PBMCs proliferate and increase CD56 expression after longer-term culture with Raji cells and RTX. Additional maturation markers were assessed to better understand the differentiation status of the CD56bright NK cells that emerge following longer-term culture with RTX. Resting CD56bright NK cells express low levels of CD16 while resting CD56dim NK cells express higher levels of CD16. The expression of CD16 by CD56dim is known to be downregulated on NK cells in response to short-term RTX activation[22, 23]. However, expression of CD16 on NK cells recovered after 7 days with the majority of CD56bright NK cells expressing CD16 (Fig. 2d, e). These cells also expressed CD57 and KIR (Fig. 2f–i) which, in the resting state, are expressed largely by CD56dim NK cells. Less pronounced changes in NK cell phenotype and some control of target cell growth were seen in the TRA control group, likely due to allogeneic NK response to Raji cells. Together, this phenotypic data suggest that RTX-activated CD56dim NK cells upregulate the expression of CD56, re-express high levels of CD16, and display other markers of mature NK cells.
T cells are required for maintaining RTX-mediated NK cell responses
NK cells were isolated from PBMC and cocultured with RTX and Raji cells for 7 days. In contrast to what was observed with NK cells in unfractionated PBMCs, RTX failed to induce CD56dim to CD56bright transition, CFSE dilution or CD16 re-expression by isolated NK cells (Fig. S2a–d). The number of NK cells remaining in the RTX group was higher than that in the TRA group but this difference was considerably less than was seen with unfractionated PBMCs (RTX to TRA NK ratio – 3.05 versus 9.42, Fig. S2e). The elimination of Raji cells by RTX was limited when isolated NK cells were used as effector cells (Fig. S2f). This suggested a cell population in PBMC was maintaining NK cell growth, viability, cytotoxicity, and phenotypic change. To identify the cellular component in PBMC supporting these changes, monocytes, B cells or T-cell subsets were depleted and remaining cells cocultured with RTX and Raji cells. Down-modulation of CD19 in response to RTX was seen within 20 h which is consistent with previous reports. T cell depletion had limited impact on elimination of target cells at 20 h (Fig. 3a, b). However after 7 days, depletion of CD3+ T cells inhibited NK cell ADCC, viability and CD16 re-expression (Fig. 3a–e). The depletion of CD3+ or CD4+ T cells significantly suppressed the CD56dim to CD56bright NK transition after 7 days (Fig. 3f, g). Suppression of the CD56dim to CD56bright NK transition was most pronounced after CD3+ depletion, but was also seen with CD4+ depletion, suggesting CD4+T cells are primarily responsible for supporting CD56dim to CD56bright NK transition but that CD8+ T cells can contribute to this process. The expression of activation markers on NK cells including CD25 and CD69 was not altered by the depletion of CD3+ T cells (Fig. S3a, b). Depletion of B cells and monocytes had minimal impact on RTX-mediated NK cell cytotoxicity, viability, or phenotypical changes after 7 days (Fig. S4). To further assess the role of T cells in RTX-mediated ADCC, isolated NK cells were cocultured with RTX and Raji cells, and autologous CD3+, CD4+ or CD8+ T cells were added back before culturing for 7 days. RTX-mediated NK ADCC was enhanced and NK cell numbers were higher when CD3+ or CD4+ T cells were added back (Fig. S5a–c). CD56dim to CD56bright transition was not induced in isolated NK cells unless CD3+ T cells, CD4+ or CD8+ T cells were added. CD3+ and CD4+ T cells triggered more CD56dim to CD56bright NK transition than did CD8+ T cells (Fig. S5d, e). CD16 recovery was only seen with CD3+ or CD4+ T cells (Fig. S5f, g). Taken together, these data demonstrate that T cells, largely CD4+ cells, are required to maintain NK cell ADCC, viability, and phenotypical changes. It is possible an allogeneic reaction between T cells and Raji cells contributed to changes in the NK cell responses. To assess this possibility, RTX was added to PBMCs enriched for autologous B cells that served as target cells for RTX and cultured for 7 days. Results in this fully autologous system were similar to those seen with Raji as target cells. RTX-mediated NK elimination of autologous B cells, NK viability, CD56dim to CD56bright transition and CD16 re-expression were suppressed by the depletion of CD3+ T cells (Fig. 4). T cell depletion did not impact NK cell activation (Fig. S3c, d).
IL2 in the immunological synapse contributes to the impact of T cells on NK cell function.
A Transwell system was used to investigate whether the impact of T cells on RTX-activated NK cells was contact dependent. When CD3+ T cells were physically separated from NK cells, NK ADCC, viability, CD56dim to CD56bright transition and CD16 recovery was significantly reduced after 7 days (Fig. 5a–d). This suggests close contact between NK cells and T cells is needed to maintain the RTX-mediated NK cell response. Importantly, this does not exclude the possibility that soluble factors secreted by T cells impact on NK cells via the immunological synapse. T cells are known to interact with NK cells via a variety of ligand-receptor pairs including IL2–IL2R, IFNg-IFNgR, CD54-LFA1 and FGFR1–CD56[25,26,27,28]. To investigate the mechanism how T cells impact RTX-mediated NK cell responses, neutralization mAbs were used to block each of these pairs. Anti-IL2 significantly inhibited NK cell ADCC, viability, CD56dim to CD56bright transition and CD16-reexpression (Fig. 5e–h). Recombinant IL2 was sufficient to maintain NK cell response without the need for T cells (Fig. 5i–l). IL2 locally produced by T cells could have higher concentration in the immunological synapse, resulting in more profound effects on NK cells. Although IL2 alone is adequate to induce NK cell functional and phenotypical changes, other ligand-receptors pairs that require cell-to-cell contact may be involved as well. Therefore, T cells impact RTX-mediated NK cell response at least partially via IL2. The need for cell–cell contact suggests this interaction may be more robust in the immunological synapse.
T cells maintain CTX-mediated NK cell responses
To assess whether the observations outlined above are limited to anti-CD20 mAb or B cells as target cells, similar studies were done evaluating changes of NK cells in response to CTX, an anti-EGFR mAb, and head and neck cancer cells. CTX induced NK cell CD56dim to CD56bright transition, maintained NK cell numbers and promoted CD16 recovery on NK cells (Fig. S6a–e) in a manner consistent with that seen with RTX. CTX-mediated effects on NK cells were dependent on the presence of CD3+ T cells and IL2 just as was seen with RTX (Fig. S6f–h). This indicates that T cells may be critical in maintaining the long-term NK cell response to a variety of mAb via IL2.
T cell activation enhances RTX-mediated NK cell function.
Studies were done to determine whether activation of T cells enhances their ability to support NK cells. T cells were depleted from PBMC. Autologous resting T cells or T cells activated by anti-CD3/CD28 beads were added back in various concentrations. Activation of T cells enhanced NK cell responses particularly at lower doses of T cells (Fig. S7). Similar results were found following addition of a bispecific anti-HLA-DR/anti-CD3 monoclonal antibody developed in our laboratory designated IDT3D (Fig. 6). 1DT3D at low concentrations enhanced NK cell ADCC, viability, CD56dim to CD56bright transition, and CD16 recovery in the presence of small numbers of T cells, in some cases less than 1% (Fig. 6). This suggests activation by bispecific anti-CD3 antibodies of small numbers of T cells in the tumor microenvironment could enhance RTX-mediated NK cell responses.
The effects of T cells on RTX-activated NK cell transcriptomics
Bulk NK cell mRNA sequencing was used to evaluate the effects of T cells on the RTX-mediated NK cell response at the transcriptional level. PBMCs (unfractionated and after T cell depletion) were cultured for 7 days with RTX and Raji cells. NK cells were then isolated from three experimental conditions: (1) 0 h, resting PBMC (NK_naive), (2) intact PBMCs (NK_PBMC), (3) T cell-depleted PBMC (NK_TCell_Dep). Transcriptomics of NK cells from the three groups were well distinguished from each other by principal component analysis (Fig. 7a), indicating they were transcriptionally different. A prime focus for analysis was on how T cells impact on RTX-mediated NK cell transcriptomics (Fig. 7b, c). The top biological processes enriched by DEGs between the NK_PBMC and NK_TCell_Dep samples included cell communication, signaling and cell division, suggesting the importance of T-NK cell interaction in NK cell proliferation. Depletion of T cells also altered the “cytokine – cytokine receptor interaction” pathway (Fig. 7d), consistent with the finding that IL2 is playing an important role. The depletion of T cells did not have a significant impact on the Fcg receptor signaling or the NK cell cytotoxicity suggesting T cells have minimal direct impact on RTX-mediated NK cell activation (Fig. 7e). This analysis further supports the experimental findings that T cells impact on RTX-mediated NK cell response mainly by enhancing NK cell viability and proliferation, not by enhancing the cytotoxic potential of the NK cells.