Figure Legends:
Figure 1: Visual abstract. Peripheral blood and gut-infiltrating lymphocyte profiles are distinct between patients with anti-CTLA-4/PD-1-associated colitis (or ipilimumab and nivolumab associated colitis “IN-COL”) and Ulcerative Colitis (UC). In gut-isolated lymphocytes, IN-COL was characterised by high proportions of T cells that were predominantly CD8+ and high levels of activated granzyme B+ mucosal associated invariant T (MAIT) cells and a low proportion of regulatory T cells (Treg). This was distinct from UC where there was a lower level of CD8+ T cell activation, no activation of MAIT cells and a high proportion of Treg. In the peripheral blood IN-COL is associated with widespread activation of CD4+ and CD8+ T cells in an “on-treatment” effect not seen in UC. Both UC and IN-COL are associated with low proportions of MAIT cells, however this was not a reliable predictor of IN-COL in a second cohort. UC but not IN-COL was associated with high levels of circulating plasmablasts.
Figure 2: In Cohort 1, treatment with combination ipilimumab and nivolumab therapy resulted in high levels of activated memory CD4+ and CD8+ T cells, independent of the presence of colitis. Data from thawed PBMC from healthy volunteers (N=17) and patients with combination ipilimumab and nivolumab therapy-associated colitis (IN-COL shaded red on graphs; N=9), active ulcerative colitis (UC N=6) and those that received combination ipilimumab and nivolumab therapy with nil autoimmune adverse events (IN-NAE shaded blue on graphs; N=11) is shown. Median and interquartile range are displayed. B/L=Baseline, TX=on treatment for 7-10 weeks. **p<0.02 by Man-Whitney test. P values <0.02 were considered significant due to Bonferroni correction for multiple comparisons. a) Patients in the IN-COL group had high levels of circulating activated memory CD45RA-HLA-DR+CD38+CD8+ T cells at the point of active colitis compared with baseline. A significant rise in this subset was also seen in the IN-NAE groups following 7-10 weeks of therapy. b) Gating strategy showing live, single lymphocytes that are CD3+8+CD45-RA-. i Healthy volunteers ii Patient with active UC iii Patient in the IN-COL group at baseline iv Results from the same IN-COL patient at the time of active colitis v Patient in the IN-NAE group at baseline vi Results from the same IN-NAE following treatment. c) At the point of colitis IN-COL patients had high proportions of circulating activated memory CD45RA-HLA-DR+CD38+ CD4+T cells. The percentage of these cells was elevated compared with baseline values. A rise in the percentage of these cells is also seen in the IN-NAE group following 7-10 weeks of treatment. d) Gating strategy showing live, single lymphocytes that are CD45RA-CD3+4+ i Healthy volunteers ii Patient with active UC iii Patient in the IN-COL group at baseline iv Results from the same IN-COL patient at the time of active colitis v Patient in the IN-NAE group at baseline vi Results from the same IN-NAE following treatment.
Figure 3: Low levels of circulating mucosal-associated invariant T (MAIT) cells at baseline in Cohort 1 patients that developed combination ipilimumab and nivolumab therapy-related colitis. Data from thawed PBMC from healthy volunteers (N=17) and patients with combination ipilimumab and nivolumab therapy-associated colitis (IN-COL shaded red; N=9), active ulcerative colitis (UC N=6) and those that received combination ipilimumab and nivolumab with nil autoimmune adverse events (IN-NAE shaded blue; N=11). Medians and interquartile range are shown. B/L=Baseline. **p<0.02 by Man-Whitney test. P values <0.03 were considered significant due to Bonferroni correction for multiple comparisons. a) Cohort 1 Patients in the IN-COL group at baseline had a paucity of MAIT cells compared with those in the IN-NAE group at baseline. Patients with active UC flare had lower circulating MAIT cell counts compared to healthy volunteers. b)MAIT cell gating strategy showing live, single lymphocytes that are CD3+8+. i Patient in IN-COL group at baseline ii Patient in IN-NAE group at baseline. c) Low circulating MAIT cells at baseline not predictive of IN-COL in Cohort 2. In Cohort 1 melanoma patients who subsequently developed dual checkpoint colitis (IN-COL shaded red; N=9) has significantly lower MAIT cell proportion at baseline, compared to healthy volunteers (N=17). Melanoma patients treated with ipilimumab and nivolumab who did not develop any autoimmune side effects did not display reduction in MAIT cells (IN-NAE shaded blue; N=11). In Cohort 2 the median baseline MAIT cell count of both the melanoma patient groups IN-COL (shaded red; N=15) and IN-NAE (shaded blue; N=9) was significantly lower than that of healthy volunteers (N=35), however, there were no further differences between the patient groups. **p<0.01 and ***p<0.001 by Mann-Whitney test. P values <0.01 were considered significant due to Bonferroni correction for multiple comparisons.
Figure 4: Active ulcerative colitis (UC) but not combination ipilimumab and nivolumab therapy-related colitis (IN-COL) is associated with high circulating plasmablasts in Cohort 1. Data from thawed PBMC from healthy volunteers (N=17) and patients with combination ipilimumab and nivolumab therapy-associated colitis (IN-COL shaded red; N=9) active ulcerative colitis (UC N=6) and those that received combination ipilimumab and nivolumab with nil autoimmune adverse events (IN-NAE shaded blue; N=11). Median and interquartile range shown. B/L=Baseline, TX=on treatment for 7-10 weeks. **p<0.01 by Man-Whitney test. P values <0.03 were considered significant due to Bonferroni correction for multiple comparisons. a) Patients with active UC had significantly elevated circulating CD19+27+38+plasmablasts compared with healthy volunteers. This change was not seen in patients treated with combination ipilimumab and nivolumab therapy, including those with IN-COL b) Plasmablast gating strategy showing live, single CD19+ lymphocytes. i Healthy volunteers ii Active UC iii IN-COL at time of colitis iv IN-NAE at week 7-10.
Figure 5: High levels of activated memory CD8+T cells and mucosal-associated invariant (MAIT) T cells and low proportion of regulatory T cells (Treg) in the gastrointestinal tissue of Cohort 3 patients with ipilimumab and nivolumab therapy-related colitis (IN-COL)­­­­­­­. Data from freshly isolated gut mononuclear cells from healthy volunteers (N=6) and patients with active ulcerative colitis (UC; N=6), those that received combination ipilimumab and nivolumab therapy with nil autoimmune adverse events (IN-NAE; N=6) and those with combination ipilimumab and nivolumab therapy-associated colitis (IN-COL; N=6), is shown. Median and interquartile range are displayed. TX= treatment *p<0.02 and **p<0.01 by Man-Whitney test. P values <0.02 were considered significant due to Bonferroni correction for multiple comparisons. a)Compared with IN-NAE, patients in the IN-COL group had i) a T cell lymphocytosis that was not explained by perturbations in the total ii) CD4+ or iv) CD8+ subset. Patients with IN-COL had elevated iii) activated memory CD4+ T cells and a greater proportion of v) activated memory CD8+ T cells compared with IN-NAE. The proportion of activated memory CD8+ T cells was greater proportion than was seen in UC. b) Compared with i) healthy volunteers, patients with ii) UC had a high proportion of FOXP3+CD25+ Treg (Quadrant 2) while low proportion of Treg were seen in iii) IN-COL. (c) Compared with the IN-NAE groups, patients with IN-COL had i) no change in total MAIT cell proportion but ii) a rise in activated and iii) granzyme B+ MAIT cells. These changes were not seen in active UC. d) Activation of MAIT cells in the gastrointestinal tissue was confirmed using a cell-specific MR1-OP5 tetramer that binds specifically to the MAIT TCR. Data from thawed frozen gut-derived mononuclear cells are shown from a patient with IN-COL (i-ii) and IN-NAE (iii-iv) and gated on live CD3+ single cells. MR1-OP5 tetramer+CD161+ T cells in ii IN-COL displayed higher levels of activation compared with those in iv IN-NAE.e) Paired data from thawed peripheral blood mononuclear cells (PBMC) and freshly isolated gut mononuclear cells from patients with combination ipilimumab and nivolumab therapy-associated colitis (IN-COL in red; N=3) and those that received combination ipilimumab and nivolumab therapy with nil autoimmune adverse events (IN-NAE in grey; N=3) is shown. Higher levels of MAIT cell activation was found in the gut tissue compared with PBMC in 2/3 IN-COL patients.
Supplementary Figure 1: Peripheral blood and gut-derived mononuclear cell (PBMC; GMNC) flow cytometric gating strategies. Data from a) thawed PBMC from a healthy volunteer is shown. Gating strategy for i) lymphocytes ii) singlets iii) Live T cells iv) CD4+ and CD8+ T cells v) CD4+ naïve (Q2), effector memory (Q4), central memory (Q1) and T cells re-expressing CD45RA (Q4); Box includes all CD45RA- memory cells vi) Highly activated CD4+CD45RA- cells in Q2 vii) CD8+ naïve (Q2), effector memory (Q4), central memory (Q1) and T cells re-expressing CD45RA (Q4); Box includes all CD45RA- memory cells viii) Highly activated CD8+CD45RA- cells in Q2. Data from b) Freshly isolated GMNC from a healthy volunteer is shown. The approach is different for GMNC given the very low proportion of CD45RA+ and naïve T cells in the gut. Gating strategy for i) lymphocytes ii) singlets iii) Live T cells iv) CD4+ and CD8+ T cells v) CD4+CD45RO+vi) Highly activated CD4+CD45RO+ cells in Q2 vii) CD8+CD45RO+vi) Highly activated CD8+CD45RO+ cells in Q2.
Supplementary Figure 2: Peripheral blood mononuclear cells (PBMC) stained with monoclonal isotype control antibodies. Data from thawed PBMC from a single healthy volunteer is shown gated on live, single T cells. a) Isotype control histograms for HLA-DR versus CD38 in i) CD8+ and ii) CD4+ T cells. Cells are also stained for CD161, CCR6, CD45RO, CD25 as a comparator for Figure 2b),d),f). b) Isotype control histograms for CD161 versus i) CCR6 and ii) TCRVα7.2. Cells are also stained for CD4, CD25, CD45RO to act as a comparator for Figure 3b).
Supplementary Figure 3: a) Validation of CD161+CCR6+ CD8+ T cells as a corelate to CD161+Vα7.2+CD8+ mucosal associated invariant T (MAIT) cells in Cohort 2. Thawed peripheral blood mononuclear cells (PBMC) from healthy volunteers (N=35; open circles) and patients with melanoma prior to treatment with ipilimumab and nivolumab (N=24) were stained with markers for MAIT cells in a single flow-cytometric assay. Of the melanoma patients some subsequently developed T cell checkpoint-inhibitor-related colitis (N=15; closed triangles) while others did not (N=9; open triangles); p=3.88x10-9 by Spearman’s correlation.b) Alternative flow cytometric gating strategies for MAIT cells. Data from thawed PBMC from single healthy volunteer is shown. As depicted, lymphocytes were gated on, live T cells, single cells, CD8+ and then gated on i) CCR6 versus CD161 or ii) T cell receptor chain Vα7.2. SSC=side scatter, FSC= forward scatter, NIR= near infra-red viability stain, H=height, A=area, TCR= T cell receptor.