Immunologic predictors of vaccine responsiveness in patients with lymphoma and CLL.

Patients with B-cell lymphomas have altered cellular components of vaccine responses due to malignancy and therapy, and the optimal timing of vaccination relative to therapy remains unknown. SARS-CoV-2 vaccines created an opportunity for new insights in vaccine timing because patients were challenged with a novel antigen across multiple phases of treatment. We studied serologic mRNA vaccine response in retrospective and prospective cohorts with lymphoma and CLL, paired with clinical and research immune parameters. Reduced serologic response was observed more frequently during active therapies, but non-response was also common within observation and post-treatment groups. Total IgA and IgM correlated with successful vaccine response. In individuals treated with CART-19, non-response was associated with reduced B and T follicular helper cells. Predictors of vaccine response varied by disease and therapeutic group, and therefore further studies of immune health during and after cancer therapies are needed to allow individualized vaccine timing.

PD-1 directed immunotherapy alters Tfh and humoral immune responses to seasonal influenza vaccine.

Anti-programmed death-1 (anti-PD-1) immunotherapy reinvigorates CD8 T cell responses in patients with cancer but PD-1 is also expressed by other immune cells, including follicular helper CD4 T cells (Tfh) which are involved in germinal centre responses. Little is known, however, about the effects of anti-PD-1 immunotherapy on noncancer immune responses in humans. To investigate this question, we examined the impact of anti-PD-1 immunotherapy on the Tfh-B cell axis responding to unrelated viral antigens. Following influenza vaccination, a subset of adults receiving anti-PD-1 had more robust circulating Tfh responses than adults not receiving immunotherapy. PD-1 pathway blockade resulted in transcriptional signatures of increased cellular proliferation in circulating Tfh and responding B cells compared with controls. These latter observations suggest an underlying change in the Tfh-B cell and germinal centre axis in a subset of immunotherapy patients. Together, these results demonstrate dynamic effects of anti-PD-1 therapy on influenza vaccine responses and highlight analytical vaccination as an approach that may reveal underlying immune predisposition to adverse events.

Vaccine-induced ICOS+CD38+ circulating Tfh are sensitive biosensors of age-related changes in inflammatory pathways.

Humoral immune responses are dysregulated with aging, but the cellular and molecular pathways involved remain incompletely understood. In particular, little is known about the effects of aging on T follicular helper (Tfh) CD4 cells, the key cells that provide help to B cells for effective humoral immunity. We performed transcriptional profiling and cellular analysis on circulating Tfh before and after influenza vaccination in young and elderly adults. First, whole-blood transcriptional profiling shows that ICOS+CD38+ cTfh following vaccination preferentially enriches in gene sets associated with youth versus aging compared to other circulating T cell types. Second, vaccine-induced ICOS+CD38+ cTfh from the elderly had increased the expression of genes associated with inflammation, including tumor necrosis factor-nuclear factor κB (TNF-NF-κB) pathway activation. Finally, vaccine-induced ICOS+CD38+ cTfh display strong enrichment for signatures of underlying age-associated biological changes. These data highlight the ability to use vaccine-induced cTfh as cellular "biosensors" of underlying inflammatory and/or overall immune health.

CD8+ T cells contribute to survival in patients with COVID-19 and hematologic cancer.

Patients with cancer have high mortality from coronavirus disease 2019 (COVID-19), and the immune parameters that dictate clinical outcomes remain unknown. In a cohort of 100 patients with cancer who were hospitalized for COVID-19, patients with hematologic cancer had higher mortality relative to patients with solid cancer. In two additional cohorts, flow cytometric and serologic analyses demonstrated that patients with solid cancer and patients without cancer had a similar immune phenotype during acute COVID-19, whereas patients with hematologic cancer had impairment of B cells and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific antibody responses. Despite the impaired humoral immunity and high mortality in patients with hematologic cancer who also have COVID-19, those with a greater number of CD8 T cells had improved survival, including those treated with anti-CD20 therapy. Furthermore, 77% of patients with hematologic cancer had detectable SARS-CoV-2-specific T cell responses. Thus, CD8 T cells might influence recovery from COVID-19 when humoral immunity is deficient. These observations suggest that CD8 T cell responses to vaccination might provide protection in patients with hematologic cancer even in the setting of limited humoral responses.

The Identity of Human Tissue-Emigrant CD8+ T Cells.

Lymphocyte migration is essential for adaptive immune surveillance. However, our current understanding of this process is rudimentary, because most human studies have been restricted to immunological analyses of blood and various tissues. To address this knowledge gap, we used an integrated approach to characterize tissue-emigrant lineages in thoracic duct lymph (TDL). The most prevalent immune cells in human and non-human primate efferent lymph were T cells. Cytolytic CD8+ T cell subsets with effector-like epigenetic and transcriptional signatures were clonotypically skewed and selectively confined to the intravascular circulation, whereas non-cytolytic CD8+ T cell subsets with stem-like epigenetic and transcriptional signatures predominated in tissues and TDL. Moreover, these anatomically distinct gene expression profiles were recapitulated within individual clonotypes, suggesting parallel differentiation programs independent of the expressed antigen receptor. Our collective dataset provides an atlas of the migratory immune system and defines the nature of tissue-emigrant CD8+ T cells that recirculate via TDL.

Convalescent plasma for pediatric patients with SARS-CoV-2-associated acute respiratory distress syndrome.

There are no proven safe and effective therapies for children who develop life-threatening complications of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Convalescent plasma (CP) has demonstrated potential benefit in adults with SARS-CoV-2, but has theoretical risks.We present the first report of CP in children with life-threatening coronavirus disease 2019 (COVID-19), providing data on four pediatric patients with acute respiratory distress syndrome. We measured donor antibody levels and recipient antibody response prior to and following CP infusion. Infusion of CP was not associated with antibody-dependent enhancement (ADE) and did not suppress endogenous antibody response. We found CP was safe and possibly efficacious. Randomized pediatric trials are needed.

Comprehensive mapping of immune perturbations associated with severe COVID-19.

Although critical illness has been associated with SARS-CoV-2-induced hyperinflammation, the immune correlates of severe COVID-19 remain unclear. Here, we comprehensively analyzed peripheral blood immune perturbations in 42 SARS-CoV-2 infected and recovered individuals. We identified extensive induction and activation of multiple immune lineages, including T cell activation, oligoclonal plasmablast expansion, and Fc and trafficking receptor modulation on innate lymphocytes and granulocytes, that distinguished severe COVID-19 cases from healthy donors or SARS-CoV-2-recovered or moderate severity patients. We found the neutrophil to lymphocyte ratio to be a prognostic biomarker of disease severity and organ failure. Our findings demonstrate broad innate and adaptive leukocyte perturbations that distinguish dysregulated host responses in severe SARS-CoV-2 infection and warrant therapeutic investigation.

Deep immune profiling of COVID-19 patients reveals distinct immunotypes with therapeutic implications.

Coronavirus disease 2019 (COVID-19) is currently a global pandemic, but human immune responses to the virus remain poorly understood. We used high-dimensional cytometry to analyze 125 COVID-19 patients and compare them with recovered and healthy individuals. Integrated analysis of ~200 immune and ~50 clinical features revealed activation of T cell and B cell subsets in a proportion of patients. A subgroup of patients had T cell activation characteristic of acute viral infection and plasmablast responses reaching >30% of circulating B cells. However, another subgroup had lymphocyte activation comparable with that in uninfected individuals. Stable versus dynamic immunological signatures were identified and linked to trajectories of disease severity change. Our analyses identified three immunotypes associated with poor clinical trajectories versus improving health. These immunotypes may have implications for the design of therapeutics and vaccines for COVID-19.

Multisystem inflammatory syndrome in children and COVID-19 are distinct presentations of SARS-CoV-2.

BACKGROUNDInitial reports from the severe acute respiratory coronavirus 2 (SARS-CoV-2) pandemic described children as being less susceptible to coronavirus disease 2019 (COVID-19) than adults. Subsequently, a severe and novel pediatric disorder termed multisystem inflammatory syndrome in children (MIS-C) emerged. We report on unique hematologic and immunologic parameters that distinguish between COVID-19 and MIS-C and provide insight into pathophysiology.METHODSWe prospectively enrolled hospitalized patients with evidence of SARS-CoV-2 infection and classified them as having MIS-C or COVID-19. Patients with COVID-19 were classified as having either minimal or severe disease. Cytokine profiles, viral cycle thresholds (Cts), blood smears, and soluble C5b-9 values were analyzed with clinical data.RESULTSTwenty patients were enrolled (9 severe COVID-19, 5 minimal COVID-19, and 6 MIS-C). Five cytokines (IFN-γ, IL-10, IL-6, IL-8, and TNF-α) contributed to the analysis. TNF-α and IL-10 discriminated between patients with MIS-C and severe COVID-19. The presence of burr cells on blood smears, as well as Cts, differentiated between patients with severe COVID-19 and those with MIS-C.CONCLUSIONPediatric patients with SARS-CoV-2 are at risk for critical illness with severe COVID-19 and MIS-C. Cytokine profiling and examination of peripheral blood smears may distinguish between patients with MIS-C and those with severe COVID-19.FUNDINGFinancial support for this project was provided by CHOP Frontiers Program Immune Dysregulation Team; National Institute of Allergy and Infectious Diseases; National Cancer Institute; the Leukemia and Lymphoma Society; Cookies for Kids Cancer; Alex's Lemonade Stand Foundation for Childhood Cancer; Children's Oncology Group; Stand UP 2 Cancer; Team Connor; the Kate Amato Foundations; Burroughs Wellcome Fund CAMS; the Clinical Immunology Society; the American Academy of Allergy, Asthma, and Immunology; and the Institute for Translational Medicine and Therapeutics.

TCF-1-Centered Transcriptional Network Drives an Effector versus Exhausted CD8 T Cell-Fate Decision.

TCF-1 is a key transcription factor in progenitor exhausted CD8 T cells (Tex). Moreover, this Tex cell subset mediates responses to PD-1 checkpoint pathway blockade. However, the role of the transcription factor TCF-1 in early fate decisions and initial generation of Tex cells is unclear. Single-cell RNA sequencing (scRNA-seq) and lineage tracing identified a TCF-1+Ly108+PD-1+ CD8 T cell population that seeds development of mature Tex cells early during chronic infection. TCF-1 mediated the bifurcation between divergent fates, repressing development of terminal KLRG1Hi effectors while fostering KLRG1Lo Tex precursor cells, and PD-1 stabilized this TCF-1+ Tex precursor cell pool. TCF-1 mediated a T-bet-to-Eomes transcription factor transition in Tex precursors by promoting Eomes expression and drove c-Myb expression that controlled Bcl-2 and survival. These data define a role for TCF-1 in early-fate-bifurcation-driving Tex precursor cells and also identify PD-1 as a protector of this early TCF-1 subset.

T follicular helper cells in human efferent lymph retain lymphoid characteristics.

T follicular helper cells (Tfh), a subset of CD4+ T cells, provide requisite help to B cells in the germinal centers (GC) of lymphoid tissue. GC Tfh are identified by high expression of the chemokine receptor CXCR5 and the inhibitory molecule PD-1. Although more accessible, blood contains lower frequencies of CXCR5+ and PD-1+ cells that have been termed circulating Tfh (cTfh). However, it remains unclear whether GC Tfh exit lymphoid tissues and populate this cTfh pool. To examine exiting cells, we assessed the phenotype of Tfh present within the major conduit of efferent lymph from lymphoid tissues into blood, the human thoracic duct. Unlike what was found in blood, we consistently identified a CXCR5-bright PD-1-bright (CXCR5BrPD-1Br) Tfh population in thoracic duct lymph (TDL). These CXCR5BrPD-1Br TDL Tfh shared phenotypic and transcriptional similarities with GC Tfh. Moreover, components of the epigenetic profile of GC Tfh could be detected in CXCR5BrPD-1Br TDL Tfh and the transcriptional imprint of this epigenetic signature was enriched in an activated cTfh subset known to contain vaccine-responding cells. Together with data showing shared TCR sequences between the CXCR5BrPD-1Br TDL Tfh and cTfh, these studies identify a population in TDL as a circulatory intermediate connecting the biology of Tfh in blood to Tfh in lymphoid tissue.

Spatial distribution and function of T follicular regulatory cells in human lymph nodes.

T follicular regulatory (Tfr) cells are a population of CD4+ T cells that express regulatory T cell markers and have been shown to suppress humoral immunity. However, the precise mechanisms and location of Tfr-mediated suppression in the lymph node (LN) microenvironment are unknown. Using highly multiplexed quantitative imaging and functional assays, we examined the spatial distribution, suppressive function, and preferred interacting partners of Tfr cells in human mesenteric LNs. We find that the majority of Tfr cells express low levels of PD-1 and reside at the border between the T cell zone and B cell follicle, with very few found in the germinal centers (GCs). Although PD-1+ Tfr cells expressed higher levels of CD38, CTLA-4, and GARP than PD-1Neg Tfr cells, both potently suppressed antibody production in vitro. These findings highlight the phenotypic diversity of human Tfr cells and suggest that Tfr-mediated suppression is most efficient at the T-B border and within the follicle, not in the GC.

Immunity against cyclin B1 tumor antigen delays development of spontaneous cyclin B1-positive tumors in p53 (-/-) mice.

We previously identified cyclin B1-specific T cells and antibodies in cancer patients with cyclin B1-positive (+) tumors and also in some healthy individuals. We also demonstrated that these responses may be important in cancer immunosurveillance by showing that vaccination against cyclin B1 prevents growth of transplantable cyclin B1(+) tumors in mice. Constitutive overexpression of cyclin B1 was determined to correlate with the lack of p53 function. This allowed us to use p53(-/-) mice as a model that better approximates human disease. These p53(-/-) mice spontaneously develop cyclin B1(+) tumors. At 5-6 weeks of age, when the mice were still healthy with no evidence of tumor, they received the cyclin B1 vaccine and were then observed for tumor growth. We demonstrate that cyclin B1 vaccination delays spontaneous cyclin B1(+) tumor growth and increases median survival of tumor-bearing p53(-/-) mice.

Healthy individuals have T-cell and antibody responses to the tumor antigen cyclin B1 that when elicited in mice protect from cancer.

We previously identified the aberrantly expressed cell cycle regulator cyclin B1 as a tumor antigen recognized by antibodies and T cells from patients with breast, lung, and head and neck cancers. Ordinarily expressed only transiently in the G2/M stage of the cell cycle in normal cells, cyclin B1 is constitutively expressed at high levels in the cytoplasm of these and many other tumor types, leading to its recognition by the cancer patient's immune system. We report here an unexpected observation that cyclin B1-specific antibody and memory CD4 and CD8 T cells are also found in many healthy individuals who have no history of cancer. Moreover, young as well as older healthy people have these responses suggesting that events other than cancer, which occur either early in life or throughout life, may lead to aberrant cyclin B1 expression and anti-cyclin B1 immunity. The role, if any, of immunity to this tumor-associated antigen is not known. We wanted to determine specifically whether immunity to cyclin B1 might be important in the immunosurveillance of cyclin B1+ tumors. We therefore tested in mice the effectiveness of vaccine-elicited anti-cyclin B1 immunity against a cyclin B1+ mouse tumor that was chosen based on our published observation that cyclin B1 overexpression is associated with the lack of p53 function. We found that cyclin B1 DNA prime-protein boost vaccine protected mice from a challenge with a tumor cell line that was established from a tumor arising in the p53(-/-) mouse that spontaneously overexpresses cyclin B1.

Cyclin B1 and other cyclins as tumor antigens in immunosurveillance and immunotherapy of cancer.

Uncontrolled cell division is an indispensable event in tumor progression, and numerous molecules involved in this process have been the focus of intense investigation in tumor biology. Cyclins, molecules that orchestrate normal cell cycle progression, are abnormally overexpressed in various human cancers. We review evidence that the immune system recognizes some abnormally expressed cyclins as tumor antigens, such as cyclin B1, and we analyze the potential of cyclins D, E, and A to serve a similar function in cancer immunosurveillance.