Prevention of respiratory virus transmission by resident memory CD8+ T cells.

An ideal vaccine both attenuates virus growth and disease in infected individuals and reduces the spread of infections in the population, thereby generating herd immunity. Although this strategy has proved successful by generating humoral immunity to measles, yellow fever and polio, many respiratory viruses evolve to evade pre-existing antibodies1. One approach for improving the breadth of antiviral immunity against escape variants is through the generation of memory T cells in the respiratory tract, which are positioned to respond rapidly to respiratory virus infections2-6. However, it is unknown whether memory T cells alone can effectively surveil the respiratory tract to the extent that they eliminate or greatly reduce viral transmission following exposure of an individual to infection. Here we use a mouse model of natural parainfluenza virus transmission to quantify the extent to which memory CD8+ T cells resident in the respiratory tract can provide herd immunity by reducing both the susceptibility of acquiring infection and the extent of transmission, even in the absence of virus-specific antibodies. We demonstrate that protection by resident memory CD8+ T cells requires the antiviral cytokine interferon-γ (IFNγ) and leads to altered transcriptional programming of epithelial cells within the respiratory tract. These results suggest that tissue-resident CD8+ T cells in the respiratory tract can have important roles in protecting the host against viral disease and limiting viral spread throughout the population.

Platinum-based chemotherapy attenuates the effector response of CD8 T cells to concomitant PD-1 blockade.

PURPOSE: Combination of chemotherapy (CT) with programmed cell death (PD)-1 blockade is a front-line treatment for lung cancer. However, it remains unknown whether and how CT affects the response of exhausted CD8 T cells to PD-1 blockade. EXPERIMENTAL DESIGN: We used the well-established mouse model of T cell exhaustion with chronic lymphocytic choriomeningitis virus (LCMV) infection to assess the effect of CT (cisplatin+pemetrexed) on T cell response to PD-1 blockade, in the absence of the impact of CT on antigen release and presentation observed in tumor models. RESULTS: When concomitantly administered with PD-1 blockade, CT affected the differentiation path of LCMV-specific CD8 T cells from stem-like to transitory effector cells, thereby reducing their expansion and production of interferon (IFN)-γ. After combination treatment, these restrained effector responses resulted in impaired viral control, compared to PD-1 blockade alone. The sequential combination strategy, where PD-1 blockade followed CT, proved to be superior to the concomitant combination, preserving the proliferative response of exhausted CD8 T cells to PD-1 blockade. Our findings suggest that the stem-like CD8 T cells themselves are relatively unaffected by CT partly because they are quiescent and maintained by slow self-renewal at the steady state. However, upon the proliferative burst mediated by PD-1 blockade, the accelerated differentiation and self-renewal of stem-like cells may be curbed by concomitant CT, ultimately resulting in impaired overall CD8 T cell effector functions. CONCLUSIONS: In a translational context, we provide a proof-of-concept to consider optimizing the timing of chemo-immunotherapy strategies for improved CD8 T cell functions.

Antibody Response to COVID-19 mRNA Vaccine in Patients With Lung Cancer After Primary Immunization and Booster: Reactivity to the SARS-CoV-2 WT Virus and Omicron Variant.

PURPOSE: To examine COVID-19 mRNA vaccine-induced binding and neutralizing antibody responses in patients with non-small-cell lung cancer (NSCLC) to SARS-CoV-2 614D (wild type [WT]) strain and variants of concern after the primary 2-dose and booster vaccination. METHODS: Eighty-two patients with NSCLC and 53 healthy volunteers who received SARS-CoV-2 mRNA vaccines were included in the study. Blood was collected longitudinally, and SARS-CoV-2-specific binding and neutralizing antibody responses were evaluated by Meso Scale Discovery assay and live virus Focus Reduction Neutralization Assay, respectively. RESULTS: A majority of patients with NSCLC generated binding and neutralizing antibody titers comparable with the healthy vaccinees after mRNA vaccination, but a subset of patients with NSCLC (25%) made poor responses, resulting in overall lower (six- to seven-fold) titers compared with the healthy cohort (P = < .0001). Although patients age > 70 years had lower immunoglobulin G titers (P = < .01), patients receiving programmed death-1 monotherapy, chemotherapy, or a combination of both did not have a significant impact on the antibody response. Neutralizing antibody titers to the B.1.617.2 (Delta), B.1.351 (Beta), and in particular, B.1.1.529 (Omicron) variants were significantly lower (P = < .0001) compared with the 614D (WT) strain. Booster vaccination led to a significant increase (P = .0001) in the binding and neutralizing antibody titers to the WT and Omicron variant. However, 2-4 months after the booster, we observed a five- to seven-fold decrease in neutralizing titers to WT and Omicron viruses. CONCLUSION: A subset of patients with NSCLC responded poorly to the SARS-CoV-2 mRNA vaccination and had low neutralizing antibodies to the B.1.1.529 Omicron variant. Booster vaccination increased binding and neutralizing antibody titers to Omicron, but antibody titers declined after 3 months. These data highlight the concern for patients with cancer given the rapid spread of SARS-CoV-2 Omicron variant.

Humoral Responses Against SARS-CoV-2 and Variants of Concern After mRNA Vaccines in Patients With Non-Hodgkin Lymphoma and Chronic Lymphocytic Leukemia.

PURPOSE: Patients with non-Hodgkin lymphoma including chronic lymphocytic leukemia (NHL/CLL) are at higher risk of severe SARS-CoV-2 infection. We investigated vaccine-induced antibody responses in patients with NHL/CLL against the original SARS-CoV-2 strain and variants of concern including B.1.167.2 (Delta) and B.1.1.529 (Omicron). MATERIALS AND METHODS: Blood from 121 patients with NHL/CLL receiving two doses of vaccine were collected longitudinally. Antibody binding against the full-length spike protein, the receptor-binding, and N-terminal domains of the original strain and of variants was measured using a multiplex assay. Live-virus neutralization against Delta, Omicron, and the early WA1/2020 strains was measured using a focus reduction neutralization test. B cells were measured by flow cytometry. Correlation between vaccine response and clinical factors was determined. RESULTS: Mean anti-SARS-CoV-2 spike immunoglobulin G-binding titers were 85-fold lower in patients with NHL/CLL compared with healthy controls, with seroconversion occurring in only 67% of patients. Neutralization titers were also lower and correlated with binding titers (P < .0001). Treatment with anti-CD20-directed therapies within 1 year resulted in 136-fold lower binding titers. Peripheral blood B-cell count also correlated with vaccine response. At 3 months from last anti-CD20-directed therapy, B-cell count ≥ 4.31/µL blood around the time of vaccination predicted response (OR 7.46, P = .04). Antibody responses also correlated with age. Importantly, neutralization titers against Delta and Omicron were reduced six- and 42-fold, respectively, with 67% of patients seropositive for WA1/2020 exhibiting seronegativity for Omicron. CONCLUSION: Antibody binding and live-virus neutralization against SARS-CoV-2 and its variants of concern including Delta and Omicron were substantially lower in patients with NHL/CLL compared with healthy vaccinees. Anti-CD20-directed therapy < 1 year before vaccination and number of circulating B cells strongly predict vaccine response.

Persistence of Virus-Specific Antibody after Depletion of Memory B Cells.

Humoral immunity is a major component of the adaptive immune response against viruses and other pathogens with pathogen-specific antibody acting as the first line of defense against infection. Virus-specific antibody levels are maintained by continual secretion of antibody by plasma cells residing in the bone marrow. This raises the important question of how the virus-specific plasma cell population is stably maintained and whether memory B cells are required to replenish plasma cells, balancing their loss arising from their intrinsic death rate. In this study, we examined the longevity of virus-specific antibody responses in the serum of mice following acute viral infection with three different viruses: lymphocytic choriomeningitis virus (LCMV), influenza virus, and vesicular stomatitis virus (VSV). To investigate the contribution of memory B cells to the maintenance of virus-specific antibody levels, we employed human CD20 transgenic mice, which allow for the efficient depletion of B cells with rituximab, a human CD20-specific monoclonal antibody. Mice that had resolved an acute infection with LCMV, influenza virus, or VSV were treated with rituximab starting at 2 months after infection, and the treatment was continued for up to a year postinfection. This treatment regimen with rituximab resulted in efficient depletion of B cells (>95%), with virus-specific memory B cells being undetectable. There was an early transient drop in the antibody levels after rituximab treatment followed by a plateauing of the curve with virus-specific antibody levels remaining relatively stable (half-life of 372 days) for up to a year after infection in the absence of memory B cells. The number of virus-specific plasma cells in the bone marrow were consistent with the changes seen in serum antibody levels. Overall, our data show that virus-specific plasma cells in the bone marrow are intrinsically long-lived and can maintain serum antibody titers for extended periods of time without requiring significant replenishment from memory B cells. These results provide insight into plasma cell longevity and have implications for B cell depletion regimens in cancer and autoimmune patients in the context of vaccination in general and especially for COVID-19 vaccines. IMPORTANCE Following vaccination or primary virus infection, virus-specific antibodies provide the first line of defense against reinfection. Plasma cells residing in the bone marrow constitutively secrete antibodies, are long-lived, and can thus maintain serum antibody levels over extended periods of time in the absence of antigen. Our data, in the murine model system, show that virus-specific plasma cells are intrinsically long-lived but that some reseeding by memory B cells might occur. Our findings demonstrate that, due to the longevity of plasma cells, virus-specific antibody levels remain relatively stable in the absence of memory B cells and have implications for vaccination.

Antibody response to SARS-CoV-2 mRNA vaccine in lung cancer patients: Reactivity to vaccine antigen and variants of concern.

PURPOSE: We investigated SARS-CoV-2 mRNA vaccine-induced binding and live-virus neutralizing antibody response in NSCLC patients to the SARS-CoV-2 wild type strain and the emerging Delta and Omicron variants. METHODS: 82 NSCLC patients and 53 healthy adult volunteers who received SARS-CoV-2 mRNA vaccines were included in the study. Blood was collected longitudinally, and SARS-CoV-2-specific binding and live-virus neutralization response to 614D (WT), B.1.617.2 (Delta), B.1.351 (Beta) and B.1.1.529 (Omicron) variants were evaluated by Meso Scale Discovery (MSD) assay and Focus Reduction Neutralization Assay (FRNT) respectively. We determined the longevity and persistence of vaccine-induced antibody response in NSCLC patients. The effect of vaccine-type, age, gender, race and cancer therapy on the antibody response was evaluated. RESULTS: Binding antibody titer to the mRNA vaccines were lower in the NSCLC patients compared to the healthy volunteers (P=<0.0001). More importantly, NSCLC patients had reduced live-virus neutralizing activity compared to the healthy vaccinees (P=<0.0001). Spike and RBD-specific binding IgG titers peaked after a week following the second vaccine dose and declined after six months (P=<0.001). While patients >70 years had lower IgG titers (P=<0.01), patients receiving either PD-1 monotherapy, chemotherapy or a combination of both did not have a significant impact on the antibody response. Binding antibody titers to the Delta and Beta variants were lower compared to the WT strain (P=<0.0001). Importantly, we observed significantly lower FRNT50 titers to Delta (6-fold), and Omicron (79-fold) variants (P=<0.0001) in NSCLC patients. CONCLUSIONS: Binding and live-virus neutralizing antibody titers to SARS-CoV-2 mRNA vaccines in NSCLC patients were lower than the healthy vaccinees, with significantly lower live-virus neutralization of B.1.617.2 (Delta), and more importantly, the B.1.1.529 (Omicron) variant compared to the wild-type strain. These data highlight the concern for cancer patients given the rapid spread of SARS-CoV-2 Omicron variant.

Dependence of CD8 T Cell Response upon Antigen Load During Primary Infection : Analysis of Data from Yellow Fever Vaccination.

A major question in immunology is what role antigen load plays in determining the size of the CD8 immune response. Is the amount of antigen important during recruitment, proliferation, and/or memory formation? Animal studies have shown that antigen is only strictly required early during activation of T cells, but the importance of antigen at later timepoints is unclear. Using data from 24 volunteers infected with the yellow fever vaccine virus (YFV), we analyzed the dependence of T cell proliferation upon viral load. We found that volunteers with high viral load initially have greater T cell responses, but by 28 days post-vaccination those with lower viral load are able to 'catch-up.' Using differential equation modeling we show that this pattern is consistent with viral load only affecting recruitment (i.e., programmed proliferation) as opposed to affecting recruitment and proliferation (i.e., antigen-dependent proliferation). A quantitative understanding of the dependence of T cell dynamics on antigen load will be of use to modelers studying not only vaccination, but also cancer immunology and autoimmune disorders.

Initial viral load determines the magnitude of the human CD8 T cell response to yellow fever vaccination.

CD8 T cells are a potent tool for eliminating intracellular pathogens and tumor cells. Thus, eliciting robust CD8 T-cell immunity is the basis for many vaccines under development. However, the relationship between antigen load and the magnitude of the CD8 T-cell response is not well-described in a human immune response. Here we address this issue by quantifying viral load and the CD8 T-cell response in a cohort of 80 individuals immunized with the live attenuated yellow fever vaccine (YFV-17D) by sampling peripheral blood at days 0, 1, 2, 3, 5, 7, 9, 11, 14, 30, and 90. When the virus load was below a threshold (peak virus load < 225 genomes per mL, or integrated virus load < 400 genome days per mL), the magnitude of the CD8 T-cell response correlated strongly with the virus load (R(2) ∼ 0.63). As the virus load increased above this threshold, the magnitude of the CD8 T-cell responses saturated. Recent advances in CD8 T-cell-based vaccines have focused on replication-incompetent or single-cycle vectors. However, these approaches deliver relatively limited amounts of antigen after immunization. Our results highlight the requirement that T-cell-based vaccines should deliver sufficient antigen during the initial period of the immune response to elicit a large number of CD8 T cells that may be needed for protection.

Towards a quantitative understanding of the within-host dynamics of influenza A infections.

Although the influenza A virus has been extensively studied, a quantitative understanding of the infection dynamics is still lacking. To make progress in this direction, we designed several mathematical models and compared them with data from influenza A infections of mice. We find that the immune response (IR) plays an important part in the infection dynamics. Both an innate and an adaptive IR are required to provide adequate explanation of the data. In contrast, regrowth of epithelial cells did not seem to be an important mechanism on the time scale of the infection. We also find that different model variants for both innate and adaptive responses fit the data well, indicating the need for additional data to allow further model discrimination.

A simple mathematical model helps to explain the immunodominance of CD8 T cells in influenza A virus infections.

Understanding immunodominance, the phenomenon of epitope-specific T cells expanding in an often distinctly hierarchical fashion, is important for the design of T-cell-based intervention strategies. Several recent studies have investigated immunodominance of H-2D(b)-restricted CD8(+) T cells specific for the nucleoprotein NP366 and acid polymerase PA224 epitopes during influenza A virus infection of C57BL/6 mice. CD8(+) T cells specific for these two epitopes are codominant during primary infection; NP366 dominates during secondary infection. While a number of explanations for this observation have been proposed, none of them can fully account for all the observed data. In this article, we use a simple mathematical model to explain the seemingly inconsistent data. We show that the dynamic interactions between CD8(+) T cells and antigen presentation lead to a situation where CD8(+) T cells are limiting during the initial response whereas antigen is limiting in the secondary response. This "numbers game" between antigen and CD8(+) T cells can reproduce the observed immunodominance of the NP336- and PA224-specific CD8(+) T cells, thereby explaining the reported experimental data.

Mathematical models of cytotoxic T-lymphocyte killing.

By killing infected host cells, cytotoxic T lymphocytes (CTL) mediate an important defense mechanism against viruses and other intracellular pathogens. Quantitative aspects of this killing process have been studied for several decades in vitro. More recently, methods have been developed to measure the timescales of CTL killing in vivo. Here, we review the estimates of kinetic rates involved in CTL killing which were obtained in these studies, and elaborate on the differences between them.

Quantifying cell turnover using CFSE data.

The CFSE dye dilution assay is widely used to determine the number of divisions a given CFSE labelled cell has undergone in vitro and in vivo. In this paper, we consider how the data obtained with the use of CFSE (CFSE data) can be used to estimate the parameters determining cell division and death. For a homogeneous cell population (i.e., a population with the parameters for cell division and death being independent of time and the number of divisions cells have undergone), we consider a specific biologically based "Smith-Martin" model of cell turnover and analyze three different techniques for estimation of its parameters: direct fitting, indirect fitting and rescaling method. We find that using only CFSE data, the duration of the division phase (i.e., approximately the S+G2+M phase of the cell cycle) can be estimated with the use of either technique. In some cases, the average division or cell cycle time can be estimated using the direct fitting of the model solution to the data or by using the Gett-Hodgkin method [Gett A. and Hodgkin, P. 2000. A cellular calculus for signal integration by T cells. Nat. Immunol. 1:239-244]. Estimation of the death rates during commitment to division (i.e., approximately the G1 phase of the cell cycle) and during the division phase may not be feasible with the use of only CFSE data. We propose that measuring an additional parameter, the fraction of cells in division, may allow estimation of all model parameters including the death rates during different stages of the cell cycle.

The rescaling method for quantifying the turnover of cell populations.

The dynamic nature of immune responses requires the development of appropriate experimental and theoretical tools to quantitatively estimate the division and death rates which determine the turnover of immune cells. A number of papers have used experimental data from BrdU and D-glucose labels together with a simple random birth-death model to quantify the turnover of immune cells focusing on HIV/SIV infections [Mohri et al. 279 (1998) 1223-1227, Hellerstein et al. 5 (1999) 83-89, Bonhoeffer et al. 164 (2000) 5049-5054, Mohri et al. 87 (2001) 1277-1287]. We show how uncertainties in the assumptions of the random birth-death model may lead to substantial errors in the parameters estimated. We then show how more accurate estimates can be obtained from the more recent CFSE data which allow to track the number of divisions each cell has undergone. Specifically, we: (i) describe a general stage-structured model of cell division where the probabilities of division and death are functions of time since the previous division; (ii) develop a rescaling method to identify invariant parameters (i.e. the ones that are independent of the specific functions describing division and death); (iii) show how these invariant parameters can be estimated, and (iv) illustrate this technique by applying it to CFSE data taken from the literature.

Estimating the precursor frequency of naive antigen-specific CD8 T cells.

The constraint of fitting a diverse repertoire of antigen specificities in a limited total population of lymphocytes results in the frequency of naive cells specific for any given antigen (defined as the precursor frequency) being below the limit of detection by direct measurement. We have estimated this precursor frequency by titrating a known quantity of antigen-specific cells into naive recipients. Adoptive transfer of naive antigen-specific T cell receptor transgenic cells into syngeneic nontransgenic recipients, followed by stimulation with specific antigen, results in activation and expansion of both donor and endogenous antigen-specific cells in a dose-dependent manner. The precursor frequency is equal to the number of transferred cells when the transgenic and endogenous responses are of equal magnitude. Using this method we have estimated the precursor frequency of naive CD8 T cells specific for the H-2D(b)-restricted GP33-41 epitope of LCMV to be 1 in 2 x 10(5). Thus, in an uninfected mouse containing approximately 2-4 x 10(7) naive CD8 T cells we estimate there to be 100-200 epitope-specific cells. After LCMV infection these 100-200 GP33-specific naive CD8 T cells divide >14 times in 1 wk to reach a total of approximately 10(7) cells. Approximately 5% of these activated GP33-specific effector CD8 T cells survive to generate a memory pool consisting of approximately 5 x 10(5) cells. Thus, an acute LCMV infection results in a >1,000-fold increase in precursor frequency of D(b)GP33-specific CD8 T cells from 2 x 10(2) naive cells in uninfected mice to 5 x 10(5) memory cells in immunized mice.