Garett Dunsmore

Biomarkers guiding the neoadjuvant use of immune-checkpoint blockers (ICB) are needed for patients with localized muscle-invasive bladder cancers (MIBC). Profiling tumor and blood samples, we found that follicular helper CD4+ T cells (TFH) are among the best therapeutic targets of pembrolizumab correlating with progression-free survival. TFH were associated with tumoral CD8 and PD-L1 expression at baseline and the induction of tertiary lymphoid structures after pembrolizumab. Blood central memory TFH accumulated in tumors where they produce CXCL13, a chemokine found in the plasma of responders only. IgG4+CD38+ TFH residing in bladder tissues correlated with clinical benefit. Finally, TFH and IgG directed against urothelium-invasive Escherichia coli dictated clinical responses to pembrolizumab in three independent cohorts. The links between tumor infection and success of ICB immunomodulation should be prospectively assessed at a larger scale.Significance:In patients with bladder cancer treated with neoadjuvant pembrolizumab, E. coli–specific CXCL13 producing TFH and IgG constitute biomarkers that predict clinical benefit. Beyond its role as a biomarker, such immune responses against E. coli might be harnessed for future therapeutic strategies.This article is highlighted in the In This Issue feature, p. 2221

Supplementary Figure from Escherichia coli–Specific CXCL13-Producing TFH Are Associated with Clinical Efficacy of Neoadjuvant PD-1 Blockade against Muscle-Invasive Bladder Cancer

Supplementary Figure from Escherichia coli–Specific CXCL13-Producing TFH Are Associated with Clinical Efficacy of Neoadjuvant PD-1 Blockade against Muscle-Invasive Bladder Cancer

Biomarkers guiding the neoadjuvant use of immune-checkpoint blockers (ICB) are needed for patients with localized muscle-invasive bladder cancers (MIBC). Profiling tumor and blood samples, we found that follicular helper CD4+ T cells (TFH) are among the best therapeutic targets of pembrolizumab correlating with progression-free survival. TFH were associated with tumoral CD8 and PD-L1 expression at baseline and the induction of tertiary lymphoid structures after pembrolizumab. Blood central memory TFH accumulated in tumors where they produce CXCL13, a chemokine found in the plasma of responders only. IgG4+CD38+ TFH residing in bladder tissues correlated with clinical benefit. Finally, TFH and IgG directed against urothelium-invasive Escherichia coli dictated clinical responses to pembrolizumab in three independent cohorts. The links between tumor infection and success of ICB immunomodulation should be prospectively assessed at a larger scale.Significance:In patients with bladder cancer treated with neoadjuvant pembrolizumab, E. coli–specific CXCL13 producing TFH and IgG constitute biomarkers that predict clinical benefit. Beyond its role as a biomarker, such immune responses against E. coli might be harnessed for future therapeutic strategies.This article is highlighted in the In This Issue feature, p. 2221

Biomarkers guiding the neoadjuvant use of immune-checkpoint blockers (ICB) are needed for patients with localized muscle-invasive bladder cancers (MIBC). Profiling tumor and blood samples, we found that follicular helper CD4+ T cells (TFH) are among the best therapeutic targets of pembrolizumab correlating with progression-free survival. TFH were associated with tumoral CD8 and PD-L1 expression at baseline and the induction of tertiary lymphoid structures after pembrolizumab. Blood central memory TFH accumulated in tumors where they produce CXCL13, a chemokine found in the plasma of responders only. IgG4+CD38+ TFH residing in bladder tissues correlated with clinical benefit. Finally, TFH and IgG directed against urothelium-invasive Escherichia coli dictated clinical responses to pembrolizumab in three independent cohorts. The links between tumor infection and success of ICB immunomodulation should be prospectively assessed at a larger scale. Significance: In patients with bladder cancer treated with neoadjuvant pembrolizumab, E. coli–specific CXCL13 producing TFH and IgG constitute biomarkers that predict clinical benefit. Beyond its role as a biomarker, such immune responses against E. coli might be harnessed for future therapeutic strategies. This article is highlighted in the In This Issue feature, p. 2221

Patients with cancer are at higher risk of severe coronavirus infectious disease 2019 (COVID-19), but the mechanisms underlying virus-host interactions during cancer therapies remain elusive. When comparing nasopharyngeal swabs from cancer and non-cancer patients for RT-qPCR cycle thresholds measuring acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in 1063 patients (58% with cancer, 89% COVID-19 ⁺ ), we found that malignant disease favors the magnitude and duration of viral RNA shedding concomitant with prolonged serum elevations of type 1 IFN that anticorrelated with anti-RBD IgG antibodies. Chronic viral RNA carriers exhibited the typical immunopathology of severe COVID-19 at the early phase of infection including circulation of immature neutrophils, depletion of non-conventional monocytes and a general lymphopenia that, however, was accompanied by a rise in plasmablasts, activated follicular T helper cells, and non-naive Granzyme B ⁺ FasL ⁺ , Eome ^high TCF-1 ^high , PD-1 ⁺ CD8 ⁺ Tc1 cells. Virus-induced lymphopenia worsened cancer-associated lymphocyte loss, and low lymphocyte counts correlated with chronic SARS-CoV-2 RNA shedding, COVID-19 severity and a higher risk of cancer-related death in the first and second surge of the pandemic. Lymphocyte loss correlated with significant changes in metabolites from the polyamine and biliary salt pathways as well as increased blood DNA from Enterobacteriaceae and Micrococcaceae gut family members in long term viral carriers. We surmise that cancer therapies may exacerbate the paradoxical association between lymphopenia and COVID-19-related immunopathology, and that the prevention of COVID-19-induced lymphocyte loss may reduce cancer-associated death.

We report significant upregulation of Galectin-9 (Gal-9) and VISTA on both CD4⁺ and CD8⁺ T cells in HIV-infected human patients. Gal-9 and VISTA expression was associated with impaired T cells effector functions. Although Gal-9 was coexpressed with other coinhibitory receptors such as TIGIT, CD160, CD39, and VISTA, it was simultaneously coexpressed with PD-1. Coexpression of Gal-9 with PD-1 was associated with a more terminally exhausted T cell phenotype in HIV-1 patients. This was marked by higher expression of EOMES, blimp1, and Glut1 in Gal-9⁺ versus Gal-9⁻ T cells, which is consistent with an exhausted T cell phenotype. Gal-9⁺ T cells exhibited the phenotype characteristics of effector T cells (CD45RA⁺, CD45RO-/lo, CD62L⁻, CD27lo) with higher T-bet expression. A positive correlation between the plasma viral load with the plasma Gal-9 levels in treatment-naive HIV patients and an inverse correlation between CD4 count with the frequency of CD4⁺Gal-9⁺ T cells were observed. Increased percentages of Gal-9⁺ T cells was evident in HIV-treated patients. Enhanced expression of Gal-9 on T cells following PMA stimulation via protein kinase C suggests persistent TCR stimulation as a potential contributing factor in Gal-9 upregulation in HIV patients. This was supported by the constant degranulation of Gal-9⁺ T cells. Moreover, CD44 clustering by Gal-9 may influence cytoskeleton rearrangement and coclustering of CD3, which likely impact initiation of signal transduction via TCR. Our preliminary data also confirm upregulation of Gal-9 on T cells in hepatitis B virus and HPV infections. These results demonstrate a novel role for Gal-9 and VISTA in HIV pathogenesis. Key Points Gal-9 and VISTA are highly expressed on T cells of HIV-infected individuals. Upregulation of Gal-9 and VISTA is associated with impaired T cell functions. CD44 clustering by Gal-9 influences cytoskeleton rearrangement of TCR.

NK cell functions are tightly regulated by the balance between the inhibitory and stimulatory surface receptors. We investigated the surface expression of galectin-9 (Gal-9) and its function in NK cells from HIV-infected individuals on antiretroviral therapy, long-term nonprogressors, and progressors compared with healthy controls. We also measured the expression of TIGIT and TIM-3 on different NK cell subpopulations and compared their functionality to Gal-9⁺ NK cells. Our data demonstrated significant upregulation of Gal-9 on NK cells in HIV-infected groups versus healthy controls. Gal-9 expression was associated with impaired expression of cytotoxic effector molecules granzyme B, perforin, and granulysin. In contrast, Gal-9 expression significantly enhanced IFN-γ expression in NK cells of HIV-1–infected individuals. We also found an expansion of TIGIT⁺ NK cells in HIV-infected individuals; however, dichotomous to Gal-9⁺ NK cells, TIGIT⁺ NK cells expressed significantly higher amounts of cytotoxic molecules but lower IFN-γ. Moreover, lower expression of cytotoxic effector molecules in Gal-9⁺ NK cells was associated with higher CD107a expression, which suggests indiscriminate degranulation. Importantly, a positive correlation between the plasma viral load and Gal-9⁺ NK cells was observed in progressors. Finally, we found that a cytokine mixture (IL-12, IL-15, and IL-18) can improve effector functions of Gal-9⁺ NK cells in HIV-infected individuals, although, such an effect was observed for Gal-9⁻ NK cells, as well. Overall, our data highlight the important role of Gal-9 in dysfunctional NK cells and, more importantly, a dichotomy for the role of Gal-9 versus TIGIT and suggest a potential new avenue for the development of therapeutic approaches.

CD71 ⁺ erythroid cells (CECs) have a wide range of immunomodulatory properties but their potential role in HIV has never been investigated before. Here, we demonstrate that CECs are abundant in the human cord blood, placental tissue and peripheral blood of pregnant mothers. We found that CECs exacerbate HIV-1 infection/replication when co-cultured with CD4 ⁺ T cells; and that pre-exposure of CD4 ⁺ T cells to CECs make them more permissible to HIV-infection. Our observations indicate how interactions of CECs with CD4 ⁺ T cells via reactive oxygen species (ROS)-dependent mechanism results in the upregulation of NF-kB, which affects the cell cycle machinery to facilitate HIV-1 replication. We found the complement receptor-1 (CD35) and the Duffy antigen receptor for chemokines (DARC) as potential HIV-target molecules are expressed significantly higher on CECs compared to mature red blood cells. However, blocking CD35 or DARC did not inhibit HIV-1 trans-infection to uninfected CD4 ⁺ T cells. We demonstrate that CECs bind to HIV-1 via CD235a and subsequently trans-infect the virus to uninfected CD4 ⁺ T cells. In addition, we found significant abundance of CECs in the blood of HIV-1 infected and anemic subjects, which enhanced HIV infection/replication in autologous CD4 ⁺ T cells similar to what we observed for the cord blood and placenta-derived CECs. In agreement, a positive correlation between the frequency of CECs with the plasma viral load in HIV-1 infected antiretroviral therapy naïve individuals was observed. In addition, we found that CECs even in the presence of Tenofovir, can trans-infect HIV-1 to CD4 ⁺ T cells. Our studies provide a novel insight into the role of CECs in HIV pathogenesis as potential contributing cells for viral persistence in the presence of antiretroviral therapy. Author summary: Despite current antiretroviral therapy, HIV-1 persists in a small pool of infected cells. A better understanding of HIV-reservoirs and influence of other non-immune cells on HIV-1 replication and transmission is a pre-requisite to the development of HIV-eradication strategies. Immature red blood cells (CD71 ⁺ erythroid cells) are physiologically abundant in newborns, cord blood, placenta and blood of pregnant women, with a wide range of immunological properties. This study demonstrates that these cells not only enhance HIV-1 infection/replication by reactive oxygen species in HIV-target cells (CD4 ⁺ T cells) but also bind to HIV and trans-infect the virus to the target cells in the presence of Tenofovir, an HIV drug. We found that these immature red blood cells are abundant in the blood of HIV-patients and anemic individuals. In addition, we observed a positive correlation between the levels of plasma viral load with the frequency of immature red blood cells in HIV-infected individuals. Therefore, our studies discover a novel role for these immature red blood in HIV pathogenesis, which encourages efforts to target these cells as adjuncts of current treatment strategies.

Survival of the allogeneic pregnancy depends on the maintenance of immune tolerance to paternal alloantigens at the fetomaternal interface. Multiple localized mechanisms contribute to the fetal evasion from the mother’s immune rejection as the fetus is exposed to a wide range of stimulatory substances such as maternal alloantigens, microbes and amniotic fluids. In this article, we demonstrate that CD71⁺ erythroid cells are expanded at the fetomaternal interface and in the periphery during pregnancy in both humans and mice. These cells exhibit immunosuppressive properties, and their abundance is associated with a Th2 skewed immune response, as their depletion results in a proinflammatory immune response at the fetomaternal interface. In addition to their function in suppressing proinflammatory responses in vitro, maternal CD71⁺ erythroid cells inhibit an aggressive allogeneic response directed against the fetus such as reduction in TNF-α and IFN-γ production through arginase-2 activity and PD-1/programmed death ligand-1 (PDL-1) interactions. Their depletion leads to the failure of gestation due to the immunological rejection of the fetus. Similarly, fetal liver CD71⁺ erythroid cells exhibit immunosuppressive activity. Therefore, immunosuppression mediated by CD71⁺ erythroid cells on both sides (mother/fetus) is crucial for fetomaternal tolerance. Thus, our results reveal a previously unappreciated role for CD71⁺ erythroid cells in pregnancy and indicate that these cells mediate homeostatic immunosuppressive/immunoregulatory responses during pregnancy.