Complete cancer regression occurs in a subset of patients following adoptive T cell therapy (ACT) of ex vivo expanded tumor-infiltrating lymphocytes (TILs). However, the low success rate presents a great challenge to broader clinical application. To provide insight into TIL-based immunotherapy, we studied a successful case of ACT where regression was observed against tumors carrying the hotspot mutation G12D in the KRAS oncogene. Four T cell receptors (TCRs) made up the TIL infusion and recognized two KRAS-G12D neoantigens, a nonamer and a decamer, all restricted by human leukocyte antigen (HLA) C*08:02. Three of them (TCR9a, 9b, and 9c) were nonamer-specific, while one was decamer-specific (TCR10). We show that only mutant G12D but not the wild-type peptides stabilized HLA-C*08:02 due to the formation of a critical anchor salt bridge to HLA-C. Therapeutic TCRs exhibited high affinities, ranging from nanomolar to low micromolar. Intriguingly, TCR binding affinities to HLA-C inversely correlated with their persistence in vivo, suggesting the importance of antigenic affinity in the function of therapeutic T cells. Crystal structures of TCR–HLA-C complexes revealed that TCR9a to 9c recognized G12D nonamer with multiple conserved contacts through shared CDR2β and CDR3α. This allowed CDR3β variation to confer different affinities via a variable HLA-C contact, generating an oligoclonal response. TCR10 recognized an induced and distinct G12D decamer conformation. Thus, this successful case of ACT included oligoclonal TCRs of high affinity recognizing distinct conformations of neoantigens. Our study revealed the potential of a structural approach to inform clinical efforts in targeting KRAS-G12D tumors by immunotherapy and has general implications for T cell-based immunotherapies.

Detection of viral DNA by cyclic GMP-AMP synthase (cGAS) is a first line of defence leading to the production of type I interferon (IFN). As HIV-1 replication is not a strong inducer of IFN, we hypothesised that an intact capsid physically cloaks viral DNA from cGAS. To test this, we generated defective viral particles by treatment with HIV-1 protease inhibitors or by genetic manipulation of gag. These viruses had defective Gag cleavage, reduced infectivity and diminished capacity to saturate TRIM5a. Importantly, unlike wild-type HIV-1, infection with cleavage defective HIV-1 triggered an IFN response in THP-1 cells that was dependent on viral DNA and cGAS. An IFN response was also observed in primary human macrophages infected with cleavage defective viruses. Infection in the presence of the capsid destabilising small molecule PF-74 also induced a cGAS-dependent IFN response. These data demonstrate a protective role for capsid and suggest that antiviral activity of capsid-and protease-targeting antivirals may benefit from enhanced innate and adaptive immunity in vivo.

CD4 and chemokine receptors mediate HIV-1 attachment and entry. They are, however, insufficient to explain the preferential viral infection of central memory T cells. Here, we identify L-selectin (CD62L) as a viral adhesion receptor on CD4+ T cells. The binding of viral envelope glycans to L-selectin facilitates HIV entry and infection, and L-selectin expression on central memory CD4+ T cells supports their preferential infection by HIV. Upon infection, the virus downregulates L-selectin expression through shedding, resulting in an apparent loss of central memory CD4+ T cells. Infected effector memory CD4+ T cells, however, remain competent in cytokine production. Surprisingly, inhibition of L-selectin shedding markedly reduces HIV-1 infection and suppresses viral release, suggesting that L-selectin shedding is required for HIV-1 release. These findings highlight a critical role for cell surface sheddase in HIV-1 pathogenesis and reveal new antiretroviral strategies based on small molecular inhibitors targeted at metalloproteinases for viral release.

150 words; Main text (excluding Methods section, references and figure legends): 14 30,544 characters including spaces; Number of figures: 6 (plus 9 supplemental) 15 16 Summary (150 words) 19Detection of viral DNA by cyclic GMP-AMP synthase (cGAS) is a first line of defence leading 20 to the production of type-I interferon (IFN). As HIV-1 is not a strong inducer of IFN we have 21 hypothesised that its capsid cloaks viral DNA from cGAS. To test this we generated defective 22 viral particles by treatment with HIV-1 protease inhibitors or by genetic manipulation of gag. 23These viruses had defective Gag cleavage, reduced infectivity and diminished capacity to 24 saturate TRIM5α. Importantly, unlike wild-type HIV-1, infection with cleavage defective HIV-1 25 triggered an IFN response in THP-1 cells and primary human macrophages that was 26 dependent on viral DNA and cGAS. Infection in the presence of the capsid destabilising small 27 molecule PF-74 also induced a cGAS-dependent IFN response. These data demonstrate a 28 protective role for capsid and suggest that antiviral activity of capsid-and protease-targeting 29 antivirals may benefit from enhanced innate and adaptive immunity in vivo. 30 31 capsid formation and activation of an IFN response that may be harnessed therapeutically in 86 vivo during PI treatment of HIV-1.87 88 Results 89 Protease inhibitor treatment of HIV-1 leads to innate immune induction in 90 macrophages 91To test the hypothesis that intact viral capsids protect HIV-1 DNA from detection by DNA 92 sensors we sought to generate defective viral particles by disrupting capsid maturation. The 93 protease inhibitor (PI) class of anti-retrovirals block the enzymatic activity of the viral 94 protease, preventing Gag cleavage and proper particle formation, as observed by electron 95 microscopy (Muller, Anders et al., 2009, Schatzl, Gelderblom et al., 1991. By producing VSV-96