Post-infusion PD-1+CD8+CAR-T cells identify patients responsive to CD19-CAR-T therapy in non-Hodgkin's lymphoma.

Chimeric antigen receptor T cell therapy (CAR-T) has revolutionized treatment for relapsed/refractory (r/r) B-cell non-Hodgkin's lymphoma (NHL). Robust biomarkers and a complete understanding of CAR-T cell function in the post-infusion phase remain limited. Here we used a 37-color spectral flow cytometry panel to perform high dimensional single cell analysis of post-infusion samples in 26 patients treated with CD28 co-stimulatory domain containing commercial CAR-T (CD28-CAR-T) for NHL and focused on computationally gated CD8+ CAR-T cells. We found that the presence of post-infusion PD-1+ CD8+ CAR-T cells at the Day 14 timepoint highly correlated with the ability to achieve complete response (CR) by 6 months. Further analysis identified multiple subtypes of CD8+ PD-1+ CAR-T cells including PD-1+ TCF1+ stem-like CAR-T cells and PD-1+ TIM3+ effector-like CAR-T cells that correlated with improved clinical outcomes such as response and progression free survival. Additionally, we identified a subset of PD-1+ CD8+ CAR+ T cells with effector-like function that was increased in patients who achieved a CR and was associated with Grade 3 or higher immune effector cell-associated neurotoxicity syndrome. Here we identified robust biomarkers of response to CD28-CAR-T and highlight the importance of PD-1 positivity in CD8+ CAR-T cells post-infusion in achieving CR.

Corrigendum: Brief research report: impact of vaccination on antibody responses and mortality from severe COVID-19.

[This corrects the article DOI: 10.3389/fimmu.2024.1325243.].

Translational and oncologic significance of tertiary lymphoid structures in pancreatic adenocarcinoma.

Pancreatic adenocarcinoma (PDAC) is an aggressive tumor with poor survival and limited treatment options. PDAC resistance to immunotherapeutic strategies is multifactorial, but partially owed to an immunosuppressive tumor immune microenvironment (TiME). However, the PDAC TiME is heterogeneous and harbors favorable tumor-infiltrating lymphocyte (TIL) populations. Tertiary lymphoid structures (TLS) are organized aggregates of immune cells that develop within non-lymphoid tissue under chronic inflammation in multiple contexts, including cancers. Our current understanding of their role within the PDAC TiME remains limited; TLS are complex structures with multiple anatomic features such as location, density, and maturity that may impact clinical outcomes such as survival and therapy response in PDAC. Similarly, our understanding of methods to manipulate TLS is an actively developing field of research. TLS may function as anti-tumoral immune niches that can be leveraged as a therapeutic strategy to potentiate both existing chemotherapeutic regimens and potentiate future immune-based therapeutic strategies to improve patient outcomes. This review seeks to cover anatomy, relevant features, immune effects, translational significance, and future directions of understanding TLS within the context of PDAC.

Brief research report: impact of vaccination on antibody responses and mortality from severe COVID-19.

Introduction: While it is established that vaccination reduces risk of hospitalization, there is conflicting data on whether it improves outcome among hospitalized COVID-19 patients. This study evaluated clinical outcomes and antibody (Ab) responses to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection/vaccines in patients with acute respiratory failure (ARF) and various comorbidities. Methods: In this single-center study, 152 adult patients were admitted to Ohio State University hospital with ARF (05/2020 - 11/2022) including 112 COVID-19-positive and 40 COVID-19-negative patients. Of the COVID-19 positive patients, 23 were vaccinated for SARS-CoV-2 (Vax), and 89 were not (NVax). Of the NVax COVID-19 patients, 46 were admitted before and 43 after SARS-CoV-2 vaccines were approved. SARS-CoV-2 Ab levels were measured/analyzed based on various demographic and clinical parameters of COVID-19 patients. Additionally, total IgG4 Ab concentrations were compared between the Vax and NVax patients. Results: While mortality rates were 36% (n=25) and 27% (n=15) for non-COVID-19 NVax and Vax patients, respectively, in COVID-19 patients mortality rates were 37% (NVax, n=89) and 70% (Vax, n=23). Among COVID-19 patients, mortality rate was significantly higher among Vax vs. NVax patients (p=0.002). The Charlson's Comorbidity Index score (CCI) was also significantly higher among Vax vs. NVax COVID-19 patients. However, the mortality risk remained significantly higher (p=0.02) when we compared COVID-19 Vax vs. NVax patients with similar CCI score, suggesting that additional factors may increase risk of mortality. Higher levels of SARS-CoV-2 Abs were noted among survivors, suggestive of their protective role. We observed a trend for increased total IgG4 Ab, which promotes immune tolerance, in the Vax vs. NVax patients in week 3. Conclusion: Although our cohort size is small, our results suggest that vaccination status of hospital-admitted COVID-19 patients may not be instructive in determining mortality risk. This may reflect that within the general population, those individuals at highest risk for COVID-19 mortality/immune failure are likely to be vaccinated. Importantly, the value of vaccination may be in preventing hospitalization as opposed to stratifying outcome among hospitalized patients, although our data do not address this possibility. Additional research to identify factors predictive of aberrant immunogenic responses to vaccination is warranted.

mRNA vaccines against SARS-CoV-2 induce divergent antigen-specific T-cell responses in patients with lung cancer.

BACKGROUND: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variant is highly transmissible and evades pre-established immunity. Messenger RNA (mRNA) vaccination against ancestral strain spike protein can induce intact T-cell immunity against the Omicron variant, but efficacy of booster vaccination in patients with late-stage lung cancer on immune-modulating agents including anti-programmed cell death protein 1(PD-1)/programmed death-ligand 1 (PD-L1) has not yet been elucidated. METHODS: We assessed T-cell responses using a modified activation-induced marker assay, coupled with high-dimension flow cytometry analyses. Peripheral blood mononuclear cells (PBMCs) were stimulated with various viral peptides and antigen-specific T-cell responses were evaluated using flow cytometry. RESULTS: Booster vaccines induced CD8+ T-cell response against the ancestral SARS-CoV-2 strain and Omicron variant in both non-cancer subjects and patients with lung cancer, but only a marginal induction was detected for CD4+ T cells. Importantly, antigen-specific T cells from patients with lung cancer showed distinct subpopulation dynamics with varying degrees of differentiation compared with non-cancer subjects, with evidence of dysfunction. Notably, female-biased T-cell responses were observed. CONCLUSION: We conclude that patients with lung cancer on immunotherapy show a substantial qualitative deviation from non-cancer subjects in their T-cell response to mRNA vaccines, highlighting the need for heightened protective measures for patients with cancer to minimize the risk of breakthrough infection with the Omicron and other future variants.

Pre-treatment inflamed tumor immune microenvironment is associated with FOLFIRINOX response in pancreatic cancer.

Introduction: Pancreatic adenocarcinoma (PDAC) is an aggressive tumor with limited response to both chemotherapy and immunotherapy. Pre-treatment tumor features within the tumor immune microenvironment (TiME) may influence treatment response. We hypothesized that the pre-treatment TiME composition differs between metastatic and primary lesions and would be associated with response to modified FOLFIRINOX (mFFX) or gemcitabine-based (Gem-based) therapy. Methods: Using RNAseq data from a cohort of treatment-naïve, advanced PDAC patients in the COMPASS trial, differential gene expression analysis of key immunomodulatory genes in were analyzed based on multiple parameters including tumor site, response to mFFX, and response to Gem-based treatment. The relative proportions of immune cell infiltration were defined using CIBERSORTx and Dirichlet regression. Results: 145 samples were included in the analysis; 83 received mFFX, 62 received Gem-based therapy. Metastatic liver samples had both increased macrophage (1.2 times more, p < 0.05) and increased eosinophil infiltration (1.4 times more, p < 0.05) compared to primary lesion samples. Further analysis of the specific macrophage phenotypes revealed an increased M2 macrophage fraction in the liver samples. The pre-treatment CD8 T-cell, dendritic cell, and neutrophil infiltration of metastatic samples were associated with therapy response to mFFX (p < 0.05), while mast cell infiltration was associated with response to Gem-based therapy (p < 0.05). Multiple immunoinhibitory genes such as ADORA2A, CSF1R, KDR/VEGFR2, LAG3, PDCD1LG2, and TGFB1 and immunostimulatory genes including C10orf54, CXCL12, and TNFSF14/LIGHT were significantly associated with worse survival in patients who received mFFX (p = 0.01). There were no immunomodulatory genes associated with survival in the Gem-based cohort. Discussion: Our evidence implies that essential differences in the PDAC TiME exist between primary and metastatic tumors and an inflamed pretreatment TiME is associated with mFFX response. Defining components of the PDAC TiME that influence therapy response will provide opportunities for targeted therapeutic strategies that may need to be accounted for in designing personalized therapy to improve outcomes.

Selective suppression of de novo SARS-CoV-2 vaccine antibody responses in patients with cancer on B cell-targeted therapy.

We assessed vaccine-induced antibody responses to the SARS-CoV-2 ancestral virus and Omicron variant before and after booster immunization in 57 patients with B cell malignancies. Over one-third of vaccinated patients at the pre-booster time point were seronegative, and these patients were predominantly on active cancer therapies such as anti-CD20 monoclonal antibody. While booster immunization was able to induce detectable antibodies in a small fraction of seronegative patients, the overall booster benefit was disproportionately evident in patients already seropositive and not receiving active therapy. While ancestral virus- and Omicron variant-reactive antibody levels among individual patients were largely concordant, neutralizing antibodies against Omicron tended to be reduced. Interestingly, in all patients, including those unable to generate detectable antibodies against SARS-CoV-2 spike, we observed comparable levels of EBV- and influenza-reactive antibodies, demonstrating that B cell-targeting therapies primarily impair de novo but not preexisting antibody levels. These findings support rationale for vaccination before cancer treatment.

Critical evaluation of an autologous peripheral blood mononuclear cell-based humanized cancer model.

The use of humanized mouse models for oncology is rapidly expanding. Autologous patient-derived systems are particularly attractive as they can model the human cancer's heterogeneity and immune microenvironment. In this study, we developed an autologous humanized mouse cancer model by engrafting NSG mice with patient-derived xenografts and infused matched peripheral blood mononuclear cells (PBMCs). We first defined the time course of xenogeneic graft-versus-host-disease (xGVHD) and determined that only minimal xGVHD was observed for up to 8 weeks. Next, colorectal and pancreatic cancer patient-derived xenograft bearing NSG mice were infused with 5x106 human PBMCS for development of the humanized cancer models (iPDX). Early after infusion of human PBMCs, iPDX mice demonstrated engraftment of human CD4+ and CD8+ T cells in the blood of both colorectal and pancreatic cancer patient-derived models that persisted for up to 8 weeks. At the end of the experiment, iPDX xenografts maintained the features of the primary human tumor including tumor grade and cell type. The iPDX tumors demonstrated infiltration of human CD3+ cells with high PD-1 expression although we observed significant intra and inter- model variability. In summary, the iPDX models reproduced key features of the corresponding human tumor. The observed variability and high PD-1 expression are important considerations that need to be addressed in order to develop a reproducible model system.

Converting Tumoral PD-L1 into a 4-1BB Agonist for Safer and More Effective Cancer Immunotherapy.

Dose-limiting toxicities are thought to temper the efficacy of single-agent 4-1BB agonists. To overcome this hurdle, in this issue of Cancer Discovery, Muik and colleagues report preclinical and clinical studies describing a first-in-class bispecific fusion protein targeting 4-1BB and PD-L1. See related article by Muik et al., p. 1248 (9).

Phase I Trial Characterizing the Pharmacokinetic Profile of N-803, a Chimeric IL-15 Superagonist, in Healthy Volunteers.

The oncotherapeutic promise of IL-15, a potent immunostimulant, is limited by a short serum t 1/2 The fusion protein N-803 is a chimeric IL-15 superagonist that has a >20-fold longer in vivo t 1/2 versus IL-15. This phase 1 study characterized the pharmacokinetic (PK) profile and safety of N-803 after s.c. administration to healthy human volunteers. Volunteers received two doses of N-803, and after each dose, PK and safety were assessed for 9 d. The primary endpoint was the N-803 PK profile, the secondary endpoint was safety, and immune cell levels and immunogenicity were measures of interest. Serum N-803 concentrations peaked 4 h after administration and declined with a t 1/2 of ∼20 h. N-803 did not cause treatment-emergent serious adverse events (AEs) or grade ≥3 AEs. Injection site reactions, chills, and pyrexia were the most common AEs. Administration of N-803 was well tolerated and accompanied by proliferation of NK cells and CD8+ T cells and sustained increases in the number of NK cells. Our results suggest that N-803 administration can potentiate antitumor immunity.

B cells imprint adoptively transferred CD8+ T cells with enhanced tumor immunity.

BACKGROUND: Adoptive T cell transfer (ACT) therapy improves outcomes in patients with advanced malignancies, yet many individuals relapse due to the infusion of T cells with poor function or persistence. Toll-like receptor (TLR) agonists can invigorate antitumor T cell responses when administered directly to patients, but these responses often coincide with toxicities. We posited that TLR agonists could be repurposed ex vivo to condition T cells with remarkable potency in vivo, circumventing TLR-related toxicity. METHODS: In this study we investigated how tumor-specific murine CD8+ T cells and human tumor infiltrating lymphocytes (TILs) are impacted when expanded ex vivo with the TLR9 agonist CpG. RESULTS: Herein we reveal a new way to reverse the tolerant state of adoptively transferred CD8+ T cells against tumors using TLR-activated B cells. We repurposed the TLR9 agonist, CpG, commonly used in the clinic, to bolster T cell-B cell interactions during expansion for ACT. T cells expanded ex vivo from a CpG-treated culture demonstrated potent antitumor efficacy and prolonged persistence in vivo. This antitumor efficacy was accomplished without in vivo administration of TLR agonists or other adjuvants of high-dose interleukin (IL)-2 or vaccination, which are classically required for effective ACT therapy. CpG-conditioned CD8+ T cells acquired a unique proteomic signature hallmarked by an IL-2RαhighICOShighCD39low phenotype and an altered metabolic profile, all reliant on B cells transiently present in the culture. Likewise, human TILs benefitted from expansion with CpG ex vivo, as they also possessed the IL-2RαhighICOShighCD39low phenotype. CpG fostered the expansion of potent CD8+ T cells with the signature phenotype and antitumor ability via empowering a direct B-T cell interaction. Isolated B cells also imparted T cells with the CpG-associated phenotype and improved tumor immunity without the aid of additional antigen-presenting cells or other immune cells in the culture. CONCLUSIONS: Our results demonstrate a novel way to use TLR agonists to improve immunotherapy and reveal a vital role for B cells in the generation of potent CD8+ T cell-based therapies. Our findings have immediate implications in the clinical treatment of advanced solid tumors.

Impaired neutralizing antibody response to COVID-19 mRNA vaccines in cancer patients.

There is currently a critical need to determine the efficacy of SARS-CoV-2 vaccination for immunocompromised patients. In this study, we determined the neutralizing antibody response in 160 cancer patients diagnosed with chronic lymphocytic leukemia (CLL), lung cancer, breast cancer, and various non-Hodgkin's lymphomas (NHL), after they received two doses of mRNA vaccines. Serum from 46 mRNA vaccinated health care workers (HCWs) served as healthy controls. We discovered that (1) cancer patients exhibited reduced neutralizing antibody titer (NT50) compared to HCWs; (2) CLL and NHL patients exhibited the lowest NT50 levels, with 50-60% of them below the detection limit; (3) mean NT50 levels in patients with CLL and NHL was ~2.6 fold lower than those with solid tumors; and (4) cancer patients who received anti-B cell therapy exhibited significantly reduced NT50 levels. Our results demonstrate an urgent need for novel immunization strategies for cancer patients against SARS-CoV-2, particularly those with hematological cancers and those on anti-B cell therapies.

Neoadjuvant presurgical PD-1 inhibition in oral cavity squamous cell carcinoma.

Oral cavity squamous cell carcinoma (OCSCC) is a prevalent surgically treated subset of head and neck cancer with frequent recurrence and poor survival. Immunotherapy has demonstrated efficacy in recurrent/metastatic head and neck cancer. However, whether antitumor responses could be fostered by neoadjuvant presurgical immunotherapy remains unclear. Using a Simon's two-stage design, we present results of a single-arm phase-II trial where 12 patients with stage II-IVA OCSCC received 3 to 4 biweekly doses of 3 mg/kg nivolumab followed by definitive surgical resection with curative intent. Presurgical nivolumab therapy in this cohort shows an overall response rate of 33% (n = 4 patients; 95% CI: 12%-53%). With a median follow up of 2.23 years, 10 out of 12 treated patients remain alive. Neoadjuvant nivolumab is safe, well-tolerated, and is not associated with delays in definitive surgical treatment in this study. This work demonstrates feasibility and safety for incorporation of nivolumab in the neoadjuvant setting for OCSCC (ClinicalTrials.gov: NCT03021993).

Impaired Neutralizing Antibody Response to COVID-19 mRNA Vaccines in Cancer Patients.

There is currently a critical need to determine the efficacy of SARS-CoV-2 vaccination for immunocompromised patients. In this study, we determined the neutralizing antibody response in 160 cancer patients diagnosed with chronic lymphocytic leukemia (CLL), lung cancer, breast cancer, and various non-Hodgkin's lymphomas (NHL), after they received two doses of mRNA vaccines. Serum from 46 mRNA vaccinated health care workers (HCWs) served as healthy controls. We discovered that (1) cancer patients exhibited reduced neutralizing antibody titer (NT 50 ) compared to HCWs; (2) CLL and NHL patients exhibited the lowest NT 50 levels, with 50-60% of them below the detection limit; (3) mean NT 50 levels in patients with CLL and NHL was ∼2.6 fold lower than those with solid tumors; and (4) cancer patients who received anti-B cell therapy exhibited significantly reduced NT 50 levels. Our results demonstrate an urgent need for novel immunization strategies for cancer patients against SARS-CoV-2, particularly those with hematological cancers and those on anti-B cell therapies.

Modeling ex vivo tumor-infiltrating lymphocyte expansion from established solid malignancies.

Adoptive transfer of tumor-infiltrating lymphocytes (TIL) elicits the regression of metastatic malignancies, yet a low proportion of patients achieve complete durable responses. The high incidence of relapse in these patients highlights the need to better understand mechanisms of tumor escape from T cell control. While melanoma has provided the foundation for developing TIL therapy, much less is known about TIL efficacy and relapse in other malignancies. We sought to investigate TIL characteristics in mouse tumors which have not been studied in this setting. Here, we expanded murine TIL ex vivo in IL-2 from fragments of multiple tumor models, including oral cavity cancer models of varying immunogenicity. Additionally, TIL was expanded from pmel-1 mice bearing B16F10 melanoma, yielding an enriched population of tumor-infiltrating TCR transgenic T cells. Murine TIL are similar to human TIL in that they express high levels of inhibitory receptors (PD-1, Tim-3, etc.) and can be expanded ex vivo in IL-2 extensively. Of clinical relevance, we draw parallels between murine and human oral cavity cancer TIL, evaluating relationships between inhibitory receptor expression and function. This platform can be used by labs even in the absence of clinical specimens or clean cell facilities and will be important to more broadly understand TIL phenotypes across many different malignancies.

Comparative analysis of antibodies to SARS-CoV-2 between asymptomatic and convalescent patients.

The SARS-CoV-2 viral pandemic has induced a global health crisis, which requires more in-depth investigation into immunological responses to develop effective treatments and vaccines. To understand protective immunity against COVID-19, we screened over 60,000 asymptomatic individuals in the Southeastern United States for IgG antibody positivity against the viral Spike protein, and approximately 3% were positive. Of these 3%, individuals with the highest anti-S or anti-RBD IgG level showed a strong correlation with inhibition of ACE2 binding and cross-reactivity against non-SARS-CoV-2 coronavirus S-proteins. We also analyzed samples from 94 SARS-CoV-2 patients and compared them with those of asymptomatic individuals. SARS-CoV-2 symptomatic patients had decreased antibody responses, ACE2 binding inhibition, and antibody cross-reactivity. Our study shows that healthy individuals can mount robust immune responses against SARS-CoV-2 without symptoms. Furthermore, IgG antibody responses against S and RBD may correlate with high inhibition of ACE2 binding in individuals tested for SARS-CoV-2 infection or post vaccination.

Differential immune signatures in the tumor microenvironment are associated with colon cancer racial disparities.

BACKGROUND: Disparities in colon cancer (CC) outcomes may be due to a more aggressive phenotype in African American patients in the setting of a decreased tumor immunity, though the precise mechanism for this result has not been well elucidated. To explore the molecular factors underlying CC disparities, we compared the immunogenomic signatures of CC from African American and European American patients. METHODS: We identified all CC patients from the publicly available Cancer Genome Atlas for whom race and survival data are available. Immunophenotype signatures were established for African American and European American patients. Comparisons were made regarding survival and a multivariable linear regression model was created to determine the association of immune cellular components with race. Differential gene expression was also assessed. RESULTS: Of the 254 patients identified, 58 (23%) were African American and 196 (77%) were European American. African American patients had a decreased progression free survival (p = 0.04). Tumors from African American patients displayed a reduced fraction of macrophages and CD8+ T cells and an increased fraction of B cells compared with tumors from European Americans. Differences persisted when controlling for sex, age, and disease stage. Immunostimulatory and immunoinhibitory gene profiles including major histocompatibility complex expression differed by race. CONCLUSIONS: Differences in the tumor immune microenvironment of African American as compared to European American CC specimens may play a role in the survival differences between the groups. These differences may provide targeted therapeutic opportunities.

Identification of human CD4+ T cell populations with distinct antitumor activity.

How naturally arising human CD4+ T helper subsets affect cancer immunotherapy is unclear. We reported that human CD4+CD26high T cells elicit potent immunity against solid tumors. As CD26high T cells are often categorized as TH17 cells for their IL-17 production and high CD26 expression, we posited these populations would have similar molecular properties. Here, we reveal that CD26high T cells are epigenetically and transcriptionally distinct from TH17 cells. Of clinical importance, CD26high and TH17 cells engineered with a chimeric antigen receptor (CAR) regressed large human tumors to a greater extent than enriched TH1 or TH2 cells. Only human CD26high T cells mediated curative responses, even when redirected with a suboptimal CAR and without aid by CD8+ CAR T cells. CD26high T cells cosecreted effector cytokines, produced cytotoxic molecules, and persisted long term. Collectively, our work underscores the promise of CD4+ T cell populations to improve durability of solid tumor therapies.