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Background T cell exhaustion, a ‘dysfunctional’ state of tumor infiltrating T cells, has been proposed as a major contributing factor for primary and acquired resistance to immune checkpoint inhibitor (ICI). Clonal diversity and composition of tumor infiltrating T lymphocytes (TILs) have been suggested to play a key role in determining the efficacy of ICI therapy. However, the relationship between T cell exhaustion, T cell clonality and the response to ICI therapy remains unclear. To elucidate this complex relationship, we performed a longitudinal analysis using serial biopsies from mouse tumor model treated with ICI and exhibiting differential responses, integrating single-cell transcriptomics, paired T cell receptor repertoire profiling, and spatial gene expression analysis.Methods We sequenced 441, 836 single-cell transcriptomes from 90 longitudinal mouse tumor biopsies and tumor draining lymph node specimens, encompassing 401,541 transcriptomes from tumor-infiltrating and peripheral immune cells. We compiled and validated our findings in a human tumor infiltrated immune cell atlas composed of 308,048 transcriptomes across 16 human cancer types. We also applied spatial gene expression analysis on end-stage mouse tumor model and analyzed publicly available colorectal tumor spatial data to investigate the distribution relationship of tumor immune compartment.Results Our findings indicated that tumors characterized by a limited TIL repertoire exhibited a predominance of terminally exhausted T cells and resistance to ICI therapy. Conversely, tumors with a more diverse T cell repertoire had increased frequencies of progenitor-exhausted T cells and showed favorable responses to ICI. Higher baseline frequency of CD4 + T helper cells and antigen-presenting cells (APCs) correlated with increased TIL repertoire diversity and likely contributed to shifts in the exhaustion landscape toward less terminally exhausted phenotypes. Using spatial gene expression analyses, we found that progenitor exhausted T cells interacting with CD4+ T helper and dendritic cells in TME, suggesting that CD4+ T-APC axis may modulate intratumoral CD8 T cell differentiation and exhaustion. We also identified similar function and differentiation trajectories of tumor-infiltrating lymphocytes in multiple human cancer types, including colorectal tumors. Combinatorial treatment of ICI with anti-CD40 agonist antibody enhanced TIL clonal diversity, shifted TILs towards progenitor exhaustion and improved anti-tumor responses.Conclusions Collectively, our study suggests that increasing clonal diversification could reshape the tumor-infiltrating T cell exhaustion landscape and represent a potential avenue for enhancing the therapeutic efficacy of immunotherapy.Ethics Approval Ethics committee of AbbVie gave ethical approval for this work.