Dynamics of CD8 T cell response and characterization of response patterns to immunotherapy. (A) C57BL/6 mice were inoculated with B16 melanoma cells and GVAX simultaneously. Treatment with anti-CTLA4 (clone 9H10) started 1 wk after inoculation to produce a heterogeneous response. Mice received therapy and were subjected to PET imaging according to the schedule shown in scheme A. (B) PET-CT images of a B6 mouse, injected with ⁸⁹Zr-PEG20-VHH X118, 9 d after inoculation of the tumor. (left) PET-CT maximum intensity projection of the mouse. (middle and right) A coronal cross section CT (middle) or PET-CT (right) image of the mouse. The images are taken through a cross section of the tumor. The box shows the tumor. (C) Mean growth of the tumor in the two cohorts, with or without therapy. Every mouse receiving therapy showed some level of response compared with the untreated cohort, albeit with significant heterogeneity, as evident from the standard deviations. Error bars represent standard deviation. (D) Comparison of the growth of tumors in two mice receiving therapy with a strong or a partial response. (E and F) for animals that received CTLA4 therapy, PET images of the tumors are shown. Tumors, as identified by CT, are delineated by the outline. The PET signals in the tumor are rendered as a heatmap. Below each image is the corresponding 3D graph, in which the z axis represents the strength of the PET signal (arbitrary units). On the right side of the PET images are shown PET signal intensities and their first derivatives (below each graph). Two (E) or three (F) different columns, as indicated with arrows, were picked, and graphs were drawn to show the local minima and maxima. The CD8 T cell signal was more homogenously distributed in mice with a strong response to CTLA4 treatment with no local minima throughout the tumor, whereas partial responders showed a more heterogeneous signal distribution with one or more local minima. Where relevant, areas with lower PET signals are indicated by arrows. The images show the dynamics of CD8 T cell throughout the tumors during 4 wk of imaging performed at 9, 16, 23, and 30 d after inoculation of the tumors. The images are representative of multiple experiments with similar results (Fig. S4; n = 15, P = 0.035).