TLR ligands are promising candidates for the development of novel vaccine adjuvants that can elicit protective immunity against emerging infectious diseases. Adjuvants have been used most frequently to increase the quantity of an immune response. However, the quality of a T cell response can be more important than its quantity. Stimulating certain pairs of TLRs induces a synergistic response in terms of activating dendritic cells and eliciting/enhancing T cell responses through clonal expansion, which increases the number of responding T cells. Here, we have found that utilizing ligands for 3 TLRs (TLR2/6, TLR3, and TLR9) greatly increased the protective efficacy of vaccination with an HIV envelope peptide in mice when compared with using ligands for only any 2 of these TLRs; surprisingly, increased protection was induced without a marked increase in the number of peptide-specific T cells. Rather, the combination of these 3 TLR ligands augmented the quality of the T cell responses primarily by amplifying their functional avidity for the antigen, which was necessary for clearance of virus. The triple combination increased production of DC IL-15 along with its receptor, IL-15Rα, which contributed to high avidity, and decreased expression of programmed death–ligand 1 and induction of Tregs. Therefore, selective TLR ligand combinations can increase protective efficacy by increasing the quality rather than the quantity of T cell responses.

Functional CD8⁺T cells in human tumors play a clear role in clinical prognosis and response to immunotherapeutic interventions. PD-1 expression in T cells involved in chronic infections and tumors such as melanoma often correlates with a state of T-cell exhaustion. Here we interrogate CD8⁺tumor-infiltrating lymphocytes (TILs) from human breast and melanoma tumors to explore their functional state. Despite expression of exhaustion hallmarks, such as PD-1 expression, human breast tumor CD8⁺TILs retain robust capacity for production of effector cytokines and degranulation capacity. In contrast, melanoma CD8⁺TILs display dramatic reduction of cytokine production and degranulation capacity. We show that CD8⁺TILs from human breast tumors can potently kill cancer cells via bi-specific antibodies. Our data demonstrate that CD8⁺TILs in human breast tumors retain polyfunctionality, despite PD-1 expression, and suggest that they may be harnessed for effective immunotherapies.

CD8 ⁺ tumor-infiltrating lymphocytes (TILs) correlate with relapse-free survival (RFS) in most cancer types, including breast cancer. However, subset composition, functional status, and spatial location of CD8 ⁺ TILs in relation to RFS in human breast tumors remain unclear. Spatial tissue analysis via quantitative immunofluorescence showed that infiltration of CD8 ⁺ T cells into cancer islands was more significantly associated with RFS than CD8 ⁺ T cell infiltration into either tumor stroma or total tumor. Localization into cancer islands within tumors is mediated by expression of the integrin CD103, which is a marker for tissue-resident memory T cells (TRMs). Analysis of fresh tumor samples revealed that CD8 ⁺ TRMs are functionally similar to other CD8 ⁺ TILs, suggesting that the basis of their protective effect is their spatial distribution rather than functional differences. Indeed, CD103 ⁺ TRMs, as compared with CD103 ^– CD8 ⁺ TILs, are enriched within cancer islands, and CD8 ⁺ TRM proximity to cancer cells drives the association of CD8 ⁺ TIL densities with RFS. Together, these findings reveal the importance of cancer island–localized CD8 ⁺ TRMs in surveillance of the breast tumor microenvironment and as a critical determinant of RFS in patients with breast cancer.

Significance: The essence of the adaptive immune response depends on the specificity of antigen receptors. This report identifies shared alpha–beta T-cell receptor (TCR) pairs from the tissues of HLA-A2 ⁺ patients with breast cancer and control donors. Using an emulsion-based RT-PCR assay, we analyzed TCR sequences from tissues ex vivo. We identified multiple TCR pairs shared between tumors, but not control samples. Although recent reports have concluded that anticancer T-cell responses depend on patient-specific mutation-associated neoantigens, this study provides evidence that T cells also recognize shared antigens. This approach has broad application to a variety of research questions where the end goal is to examine T-cell repertoires and/or identify T-cell antigens.

CD8 ⁺ tumor-infiltrating lymphocytes (TILs) are associated with improved survival in triple-negative breast cancer (TNBC) yet have no association with survival in estrogen receptor–positive (ER ⁺ ) BC. The basis for these contrasting findings remains elusive. We identified subsets of BC tumors infiltrated by CD8 ⁺ T cells with characteristic features of exhausted T cells (T _EX ). Tumors with abundant CD8 ⁺ T _EX exhibited a distinct tumor microenvironment marked by amplified interferon-γ signaling–related pathways and higher programmed death ligand 1 expression. Paradoxically, higher levels of tumor-infiltrating CD8 ⁺ T _EX associated with decreased overall survival of patients with ER ⁺ BC but not patients with TNBC. Moreover, high tumor expression of a CD8 ⁺ T _EX signature identified dramatically reduced survival in premenopausal, but not postmenopausal, patients with ER ⁺ BC. Finally, we demonstrated the value of a tumor T _EX signature score in identifying high-risk premenopausal ER ⁺ BC patients among those with intermediate Oncotype DX Breast Recurrence Scores. Our data highlight the complex relationship between CD8 ⁺ TILs, interferon-γ signaling, and ER status in BC patient survival. This work identifies tumor-infiltrating CD8 ⁺ T _EX as a key feature of reduced survival outcomes in premenopausal patients with early-stage ER ⁺ BC.

Objective: To determine whether myeloid cells (such as granulocytes) present in the synovial fluid (SF) of arthritic joints have an impact on adaptive immunity. Specifically, we investigated the effects of SF cells harvested from the joints of mice with proteoglycan‐induced arthritis (PGIA), on dendritic cell (DC) maturation and antigen‐specific T cell proliferation.Methods: We monitored DC maturation (MHCII and CD86 expression) by flow cytometry upon coculture of DCs with SF cells or spleen myeloid cells from mice with PGIA. The effects of these myeloid cells on T cell proliferation were studied using T cells purified from PG‐specific T cell receptor (TCR)–transgenic (Tg) mice. Phenotype analysis of myeloid cells was performed by immunostaining, reverse transcription–polymerase chain reaction, Western blotting, and biochemical assays.Results: Inflammatory SF cells significantly suppressed the maturation of DCs upon coculture. PG‐TCR–Tg mouse T cells cultured with antigen‐loaded DCs showed dramatic decreases in proliferation in the presence of SF cells. Spleen myeloid cells from arthritic mice did not have suppressive effects. SF cells were unable to suppress CD3/CD28‐stimulated proliferation of the same T cells, suggesting a DC‐dependent mechanism. SF cells exhibited all of the characteristics of myeloid‐derived suppressor cells (MDSCs) and exerted suppression primarily through the production of nitric oxide and reactive oxygen species by granulocyte‐like cells.Conclusion: SF in the joints of mice with PGIA contains a population of granulocytic MDSCs that potently suppress DC maturation and T cell proliferation. These MDSCs have the potential to limit the expansion of autoreactive T cells, thus breaking the vicious cycle of autoimmunity and inflammation.