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Cancer immunotherapies have the potential to induce durable anti-tumor immune responses; however the efficacy of these treatments varies among patients and partially depends on the complex interplay between malignant cells and non-malignant cells within the tumor microenvironment. In the tumor microenvironment, T cells play a crucial role in eliminating tumor cells and orchestrating anti-tumor immune responses; in contrast immunosuppressive cell types can support tumor progression. Here we harnessed recent advances in single-cell multiomics that allow high-resolution functional characterization of intratumoral immune cells to improve our understanding of their dynamic relationships. Specifically, we utilized a One Solution Workflow to dissociate a solid tumor followed by in-depth dissection of the immune composition of the tumor microenvironment using both flow cytometry and the BD Rhapsody™ Single-Cell Analysis System. We characterized the cellular composition of tumor microenvironment using flow cytometry. Importantly, by examining the expression of mRNA and surface protein markers at single-cell resolution, we unraveled the complexities of the tumor microenvironment, elucidated cellular heterogeneity and identified cell-type specific gene signatures. We propose this One Solution Workflow can be adopted to understand the transcriptional and proteomic phenotypes of tumor infiltrating leukocytes for development of novel immune therapeutic strategies for cancer. For Research Use Only. Not for use in diagnostic or therapeutic procedures. Class 1 Laser Product. BD-23279 (v1.0) 1120 BD, the BD Logo and Rhapsody are trademarks of Becton, Dickinson and Company or its affiliates. © 2020 BD. All rights reserved.

Cancer treatment has been revolutionized with the development of immunomodulatory therapies. While these therapies have primarily focused on enhancing T-cell responses, there has been interest in harnessing the potential of other cytotoxic cells such as Natural Killer (NK) cells. Similar to NK cells, innate lymphoid cells (ILCs) may offer another target of these immunotherapy approaches. However, there is a need for better understanding of these recently described cells. In this study, we developed a comprehensive approach to further refine the signatures of human circulating ILC subsets. Total ILCs were enriched using the BD FACSAria™ Fusion cell sorter and processed for downstream single-cell multiomic characterization. BD® AbSeq reagents and a targeted BD Rhapsody™ Immune Response Panel enabled simultaneous detection of 42 proteins and 399 genes using the BD Rhapsody™ Single-Cell Analysis System. Differential protein and gene expression analysis in addition to combinatorial expression of CD294 and CD117 confirmed 3 conventional ILC populations as well as the signatures of 3 distinct subsets within ILC1. This discovery approach provided information about relative expression of a small selection of proteins or surface marker-coding genes that enable the discrimination of these ILC subsets. These data were used to design high-parameter flow cytometry panels for high-throughput analysis. ILC subsets differentially distributed across donors were detected and defined using unsupervised computational analysis confirming the result of multiomic analysis. For Research Use Only. Class 1 laser product. BD, the BD Logo, and Rhapsody are trademarks of Becton, Dickinson and Company or its affiliates. © 2019 BD. All rights reserved.

Human regulatory T cells (Treg) are able to actively suppress autoreactive immune responses. Phenotypically, they are broadly characterized as CD4+, CD25+, CD127(lo/⁻) and FoxP3+. CD45RA can be used to further differentiate the population into naïve (CD45RA(+)) and induced (CD45RA⁻) Treg. The functional potential of Treg is routinely determined by assessing their ability to suppress T cell function in 3-5day proliferation assays. Since Treg are being explored for therapeutic use, a short-term functional assay could serve as a valuable tool for evaluating the potency of Treg. Therefore, an assay designed to measure Treg suppression of activation marker expression by responder T cells in 7 to 20h has been examined in this report. Using flow cytometry, expression of CD69 and CD154 on T cells, in response to stimulation with CD3/CD28 beads, was used as a measure of activation in the assay. Treg from healthy volunteers were sorted as CD4+CD25+CD127(lo/⁻)CD45RA+ cells with a BD FACSAria™ II. The highly purified Treg were then expanded in vitro and their function was assessed in short term activation marker suppression assays using autologous PBMC as responder cells. The data suggest that this short term suppression assay could be a reliable surrogate for assessing Treg functional potential.

We compared TLR responsiveness in PBMC from HIV-1-infected and uninfected individuals using the TLR agonists: TLR7 (3M-001), TLR8 (3M-002), and TLR7/8 (3M-011). Activation and maturation of plasmacytoid dendritic cells (pDC) were measured by evaluating CD86, CD40, and CD83 expression and myeloid dendritic cell (mDC) activation was measured by evaluating CD40 expression. All agonists tested induced activation and maturation of pDC in PBMC cultures of cells from HIV+ and HIV- individuals. The TLR7 agonist induced significantly less pDC maturation in cells from HIV+ individuals. Quantitative assessment of secreted IFN-alpha and pro-inflammatory cytokines at the single cell level showed that pDC from HIV+ individuals stimulated with TLR7 and TLR7/8 induced IFN-alpha. TLR8 and TLR7/8 agonists induced IL-12 and COX-2 expression in mDC from HIV+ and HIV- individuals. Understanding pDC and mDC activation and maturation in HIV-1 infection could lead to more rational development of immunotherapeutic strategies to stimulate the adaptive immune response to HIV-1. COL0012444

The overall prevalence with which endogenous tumor Ags induce host T cell responses is unclear. Even when such responses are detected, they do not usually result in spontaneous remission of the cancer. We hypothesized that this might be associated with a predominant phenotype and/or cytokine profile of tumor-specific responses that is different from protective T cell responses to other chronic Ags, such as CMV. We detected significant T cell responses to CEA, HER-2/neu, and/or MAGE-A3 in 17 of 21 breast cancer patients naive to immunotherapy. The pattern of T cell cytokines produced in response to tumor-associated Ags (TAAs) in breast cancer patients was significantly different from that produced in response to CMV or influenza in the same patients. Specifically, there was a higher proportion of IL-2-producing CD8+ T cells, and a lower proportion of IFN-γ-producing CD4+ and/or CD8+ T cells responding to TAAs compared with CMV or influenza Ags. Finally, the phenotype of TAA-responsive CD8+ T cells in breast cancer patients was almost completely CD28+CD45RA− (memory phenotype). CMV-responsive CD8+ T cells in the same patients were broadly distributed among phenotypes, and contained a high proportion of terminal effector cells (CD27−CD28−CD45RA+) that were absent in the TAA responses. Taken together, these results suggest that TAA-responsive T cells are induced in breast cancer patients, but those T cells are phenotypically and functionally different from CMV- or influenza-responsive T cells. Immunotherapies directed against TAAs may need to alter these T cell signatures to be effective.

This chapter describes the optimized methods for cytokine flow cytometry (CFC) that offer increased throughput and robustness. The basic principle of the CFC assay is that whole blood or peripheral blood mononuclear cells (PBMCs) are activated with a specific antigen in the presence of a secretion inhibitor such as brefeldin A (BFA) or monensin for a short duration. The samples in the plates can be acquired using a multiwell-plate loader connected to the flow cytometer. Dynamic gating strategies that account for sample-to-sample staining variability can simplify data analysis and improve the reproducibility of the results. The procedural details discussed in the chapter include recommendations for sample type and handling, choice of antigen(s) and plates, and the gating strategy for this improved CFC format using cytomegalovirus (CMV) pp65 peptide mix as a model antigen. Gating of flow cytometry data for analysis is a frequent source of assay variation, especially in rare-event assays such as CFC where antigen-specific responses may be as low as 0.1%. In situations where large numbers of samples need to be evaluated, e.g., in vaccine immune response-monitoring studies, it is desirable to minimize time and errors that may occur in assay setup and processing. The authors have optimized the process of using lyophilized antigens and lyophilized antibody cocktails for CFC assays of both PBMC and whole blood.

The ability to cryopreserve lymphocytes in peripheral blood mononuclear cells (PBMC) to retain their function after thawing is critical to the analysis of cancer immunotherapy studies. We evaluated a variety of cryopreservation strategies with the aim of developing an optimized protocol for freezing and thawing PBMC to retain viability and function. We determined several factors which do not affect cell viability after cryopreservation such as shipping frozen samples on dry ice, the length of time and speed at which samples are washed and centrifuged after thawing, and the number of cells frozen per container. Different media additives, however, did impact the viability of the cells after thawing. There was a significant reduction in the viability of the cells after freezing when using human AB serum compared to all other additives tested (p

CD4(+)CD8(dim) T cells represent a minor subset of the total CD3(+) T cell population in peripheral blood. Although transient and persistent expansions of these cells have been reported in both healthy and diseased individuals, the functional properties of the CD4(+)CD8(dim) population are largely unknown. In this study, we examined antigen-specific cytokine and proliferative responses of the CD4(+)CD8(dim) subset. In whole blood cultures stimulated with the viral antigens HCMV and HIV-1, a significant fraction of the CD4(+)CD8(dim) subset exhibited cytokine expression and proliferation in response to antigen activation. Typically, the CD4(+)CD8(dim) population contained two- to eightfold higher frequencies of antigen-specific cytokine producing cells than the CD4(+)CD8(-) population. Phenotypic analysis of the cytokine expressing CD4(+)CD8(dim) population indicated that these cells are memory T cells, with a high frequency of this population expressing the cytotoxic markers CD56 and perforin. Furthermore, the CD4(+)CD8(dim) cytokine responses to CMV were shown to be MHC class II dependent. Significantly, purified CD4(+)CD8(dim) T cells were found to possess higher CMV-specific cytotoxic activity than purified CD4(+)CD8(-) T cells in a standard (51)Cr-release CTL assay. Thus, CD4(+)CD8(dim) T cells appear to be MHC class II dependent, are capable of cytolytic effector activity, and are highly enriched within the CD4(+) cell populations specific for HCMV and HIV-1.

Antigen-specific CD8+T cells with cytotoxic activity are often critical in immune responses to infectious pathogens. To determine whether gamma interferon (IFN-γ) expression is a surrogate marker for cytotoxic T lymphocytes (CTL), human cytomegalovirus-specific CTL responses were correlated with CD8+T-cell IFN-γ expression determined by cytokine flow cytometry. A strong positive correlation was observed between specific lysis of peptide-pulsed targets in a51Cr release assay and frequencies of peptide-activated CD8+T cells expressing IFN-γ at 6 h (r2= 0.72) or 7 days (r2= 0.91). Enumeration of responding cells expressing perforin, another marker associated with CTL, did not improve this correlation. These results demonstrate that IFN-γ expression can be a functional surrogate for identification of CTL precursor cells.

Recent studies have demonstrated that therapies targeting the innate immune system have the potential to provide transient, non-specific protection from a variety of infectious organisms; however, the potential of enhancing the efficacy of such treatments using nano-scale delivery platforms requires more in depth evaluation. As such, we employed a nanolipoprotein (NLP) platform to enhance the efficacy of innate immune agonists. Here, we demonstrate that the synthetic Toll-like receptor (TLR) agonists monophosphoryl lipid A (MPLA) and CpG oligodeoxynucleotides (CpG) can be readily incorporated into NLPs. Conjugation of MPLA and CpG to NLPs (MPLA:NLP and CpG:NLP, respectively) significantly enhanced their immunostimulatory profiles both in vitro and in vivo compared to administration of agonists alone, as evidenced by significant increases in cytokine production, cell surface expression of activation markers, and upregulation of immunoregulatory genes. Importantly, enhancement of cytokine production by agonist conjugation to NLPs was also observed in primary human dendritic cells. Furthermore, BALB/c mice pretreated with CpG:NLP constructs survived a lethal influenza challenge whereas pretreatment with CpG alone had no effect on survival.

Chronic immune activation despite long-term therapy poses an obstacle to immune recovery in HIV infection. The role of antigen presenting cells (APCs) in chronic immune activation during HIV infection remains to be fully determined. APCs, the frontline of immune defense against pathogens, are capable of distinguishing between pathogens and non-pathogenic, commensal bacteria. We hypothesized that HIV infection induces dysfunction in APC immune recognition and response to some commensal bacteria and that this may promote chronic immune activation. Therefore we examined APC inflammatory cytokine responses to commensal lactobacilli. We found that APCs from HIV-infected patients produced an enhanced inflammatory response to Lactobacillus plantarum WCFS1 as compared to APCs from healthy, HIV-negative controls. Increased APC expression of TLR2 and CD36, signaling through p38-MAPK, and decreased expression of MAP kinase phosphatase-1 (MKP-1) in HIV infection was associated with this heightened immune response. Our findings suggest that chronic HIV infection enhances the responsiveness of APCs to commensal lactobacilli, a mechanism that may partly contribute to chronic immune activation. © 2013 Nagy et al.

The model of solid tumors as monolithic entities under the control of a handful of driver genes which seemed to dominate the general perception of cancer for many years is increasingly being replaced by that of highly diverse ecosystems of cells. Multiple tumor subpopulations, as well as attending non-cancerous cells such as fibroblasts, endothelial cells, and cells from the immune compartment populate this ecosystem, communicate with each other and all influence clinically relevant decision points throughout tumorigenesis. Progress in analyzing and characterizing this highly heterogeneous, complex “society of cells” has been remarkably enhanced using the strengths of flow cytometry. A rather comprehensive characterization of individual cells within a population, known as deep phenotyping, is becoming possible with recent advances in multi-parametric staining, collection, and analysis of solid tissue derived cells. This technology is enabling researchers to identify multiple targets within a single sample more efficiently than with more traditional methods. We have developed a process whereby single cells are liberated from solid tumors through a combination of mechanical and enzymatic treatments and then interrogated by flow cytometry using deep phenotyping. Our chosen marker panels include targets (CD24, CD44, CD49f, EpCAM, CD166, CD133, CD184, HER2/Neu) which have been used to investigate properties relevant for cancer stem cells (CSC), Endothelial Mesenchymal Transition (EMT) and Tumor Microenvironmental (TME) processes but options are built into the design to accommodate less well characterized targets such as GD2, CD73, Notch receptors, EphB2, and c-Met. We have identified discrete subpopulations in breast cancer patient-derived xenograft (PDX) tumors in mice, demonstrating consistent and reproducible results which constitute a distinct immunophenotypic fingerprint for every model. Building on the experience with PDX derived biopsies and adding markers targeting the immune system we applied our work flow to clinical breast cancer tissue. Our results demonstrate that the multi-dimensional analyses enabled by a surface marker panel reveals differences in highly characterized subpopulations, some at less than 1% of the total population, that remain hidden by more conventional assessment methods. Citation Format: Friedrich Hahn, Eileen Snowden, Warren Porter, Mitchell Ferguson, Shannon Dillmore, Aaron Middlebrook, Shahryar Niknam, Peter Llontop, Smita Ghanekar, Rainer Blaesius. Deep phenotyping of dissociated cells from PDX model solid tumors and human breast tumors using flow cytometry. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B11.

Purpose: We aim to characterize VTX-2337, a novel Toll-like receptor (TLR) 8 agonist in clinical development, and investigate its potential to improve monoclonal antibody–based immunotherapy that includes the activation of natural killer (NK) cells. Experimental Design: HEK-TLR transfectants were used to compare the selectivity and potency of VTX-2337, imiquimod, CpG ODN2006, and CL075. The ability of VTX-2337 to induce cytokine and chemokine production from human peripheral blood mononuclear cells (PBMC) and activation of specific immune cell subsets was examined. The potential for VTX-2337 to activate NK cell activity through direct and indirect mechanisms was also investigated. Finally, we tested the potential for VTX-2337 to augment antibody-dependent cell-mediated cytotoxicity (ADCC), especially in individuals with low-affinity FcγR3A single-nucleotide polymorphism (SNP). Results: VTX-2337 selectively activates TLR8 with an EC50 of about 100 nmol/L and stimulates production of TNFα and interleukin (IL)-12 from monocytes and myeloid dendritic cells (mDC). VTX-2337 stimulates IFNγ production from NK cells and increases the cytotoxicity of NK cells against K562 and ADCC by rituximab and trastuzumab. Effects of VTX-2337 on NK cells were, in part, from direct activation as increased IFNγ production and cytotoxic activity were seen with purified NK cells. Finally, VTX-2337 augments ADCC by rituximab in PBMCs with different FcγR3A genotypes (V/V, V/F, and F/F at position 158). Conclusions: VTX-2337 is a novel small-molecule TLR8 agonist that activates monocytes, DCs, and NK cells. Through the activation of NK cells, it has the potential to augment the effectiveness of monoclonal antibody treatments where a polymorphism in FcγR3A limits clinical efficacy. Clin Cancer Res; 18(2); 499–509. ©2011 AACR.

Background Single-cell assays of immune function are increasingly used to monitor T cell responses in immunotherapy clinical trials. Standardization and validation of such assays are therefore important to interpretation of the clinical trial data. Here we assess the levels of intra-assay, inter-assay, and inter-operator precision, as well as linearity, of CD8+ T cell IFNγ-based ELISPOT and cytokine flow cytometry (CFC), as well as tetramer assays. Results Precision was measured in cryopreserved PBMC with a low, medium, or high response level to a CMV pp65 peptide or peptide mixture. Intra-assay precision was assessed using 6 replicates per assay; inter-assay precision was assessed by performing 8 assays on different days; and inter-operator precision was assessed using 3 different operators working on the same day. Percent CV values ranged from 4% to 133% depending upon the assay and response level. Linearity was measured by diluting PBMC from a high responder into PBMC from a non-responder, and yielded R2 values from 0.85 to 0.99 depending upon the assay and antigen. Conclusion These data provide target values for precision and linearity of single-cell assays for those wishing to validate these assays in their own laboratories. They also allow for comparison of the precision and linearity of ELISPOT, CFC, and tetramer across a range of response levels. There was a trend toward tetramer assays showing the highest precision, followed closely by CFC, and then ELISPOT; while all three assays had similar linearity. These findings are contingent upon the use of optimized protocols for each assay.

Background Monocyte-derived-dendritic-cells (MDDC) are the major DC type used in vaccine-based clinical studies for a variety of cancers. In order to assess whether in vitro differentiated MDDC from cryopreserved PBMC of cancer patients are functionally distinct from those of healthy donors, we compared these cells for their expression of co-stimulatory and functional markers. In addition, the effect of cryopreservation of PBMC precursors on the quality of MDDC was also evaluated using samples from healthy donors. Methods Using flow cytometry, we compared normal donors and cancer patients MDDC grown in the presence of GM-CSF+IL-4 (immature MDDC), and GM-CSF+IL-4+TNFα+IL-1β+IL-6+PGE-2 (mature MDDC) for (a) surface phenotype such as CD209, CD83 and CD86, (b) intracellular functional markers such as IL-12 and cyclooxygenase-2 (COX-2), (c) ability to secrete IL-8 and IL-12, and (d) ability to stimulate allogeneic and antigen-specific autologous T cells. Results Cryopreservation of precursors did affect MDDC marker expression, however, only two markers, CD86 and COX-2, were significantly affected. Mature MDDC from healthy donors and cancer patients up-regulated the expression of CD83, CD86, frequencies of IL-12+ and COX-2+ cells, and secretion of IL-8; and down-regulated CD209 expression relative to their immature counterparts. Compared to healthy donors, mature MDDC generated from cancer patients were equivalent in the expression of nearly all the markers studied and importantly, were equivalent in their ability to stimulate allogeneic and antigen-specific T cells in vitro. Conclusion Our data show that cryopreservation of DC precursors does not significantly affect the majority of the MDDC markers, although the trends are towards reduced expression of co-stimulatory makers and cytokines. In addition, monocytes from cryopreserved PBMC of cancer patients can be fully differentiated into mature DC with phenotype and function equivalent to those derived from healthy donors.

Dendritic cells (DCs) are believed to regulate T cell-mediated immunity primarily by directing differentiation of naive T cells. Here, we show that a large fraction of CD4+ memory cells produce IL-10 within the first hours after interaction with plasmacytoid DCs (PDCs). In contrast, CD11c+ DCs induce IFN-γ and little IL-10. IL-10–secreting T cells isolated after 36 hours of culture with PDCs suppressed antigen-induced T-cell proliferation by an IL-10–dependent mechanism, but were distinct from natural and type 1 regulatory T cells. They proliferated strongly and continued to secrete IL-10 during expansion with PDCs, and after restimulation with immature monocyte-derived DCs or CD11c+ DCs. The IL-10–producing T cells acquired the ability to secrete high levels of IFN-γ after isolation and subsequent coculture with PDCs or CD11c+ DCs. Compared to CD11c+ DCs, PDCs were superior in their ability to selectively expand T cells that produced cytokines on repeated antigenic challenge. The DC-dependent differences in cytokine profiles were observed with viral recall antigen or staphylococcal enterotoxin B and were independent of extracellular type I interferon or IL-10. Our results show that DCs can regulate memory responses and that PDCs rapidly induce regulatory cytokines in effector T cells that can suppress bystander activity.

More precise quantitation of cellular immune responses has become possible with the advent of single-cell assays of immune function, such as cytokine flow cytometry, enzyme-linked immunospot (ELISPOT), and MHC-peptide multimers. Cytokine flow cytometry is an attractive technique because it allows the detection of responses to whole antigens without regard to MHC restriction, while also collecting additional information on responding cells via multiparameter flow cytometry. In this review, we compare cytokine flow cytometry with other assays of immune function, summarize some of that data that have been collected in various disease states using cytokine flow cytometry, and describe some methodological improvements designed to increase the robustness, throughput, and information content of this technique. We hypothesize that a new generation of automated cytokine flow cytometry assays will allow elucidation of the correlates of protection for diseases involving cellular immunity, through application of these assays in more and large clinical trials.

To study the dynamics of cytomegalovirus (CMV) immunity in healthy immunocompetent hosts, interferon-gamma-producing CD4 and CD8 T cell responses in the presence or absence of CMV antigens were measured from 15 CMV-seropositive donors and 13 CMV-seronegative donors. Cytokine responses in the absence of antigen were significantly higher in CMV-seropositive donors. Also, a disproportionate number of CD69(+) cells isolated ex vivo from CMV-seropositive donors were specific for CMV, suggesting recent reactivation in vivo. To examine changes in cellular responses over time, 10 seropositive donors were tested over a 6-month period. About half of the donors showed significant variability over time, but staphylococcal enterotoxin B responses remained relatively constant. These findings suggest that CMV can present a considerable and recurrent burden to the human immune system. By understanding the normal dynamics of CMV responses over time, it may be possible to better identify aberrant responses to CMV in immunocompromised hosts.

The effect of highly active antiretroviral therapy (HAART) on HIV-specific CD4 T cell proliferation in long-term HIV-infected individuals was studied. Subjects receiving treatment for over a year were compared with individuals not receiving therapy. The absolute number of HIV-specific memory CD4 T cells proliferating in response to HIV antigen was substantially higher in untreated subjects than in those on HAART. A decrease in HIV-specific memory CD4 T cells could explain the rebound in HIV replication after the termination of HAART.

Processing of exogenous protein Ags by APC leads predominantly to presentation of peptides on class II MHC and, thus, stimulation of CD4+ T cell responses. However, “cross-priming” can also occur, whereby peptides derived from exogenous Ags become displayed on class I MHC molecules and stimulate CD8+ T cell responses. We compared the efficiency of cross-priming with exogenous proteins to use of peptide Ags in human whole blood using a flow cytometry assay to detect T cell intracellular cytokine production. CD8+ T cell responses to whole CMV proteins were poorly detected (compared with peptide responses) in most CMV-seropositive donors. Such responses could be increased by using higher doses of Ag than were required to achieve maximal CD4+ T cell responses. A minority of donors displayed significantly more efficient CD8+ T cell responses to whole protein, even at low Ag doses. These responses were MHC class I-restricted and dependent upon proteosomal processing, indicating that they were indeed due to cross-priming. The ability to efficiently cross-prime was not a function of the number of dendritic cells in the donor’s blood. Neither supplementation of freshly isolated dendritic cells nor use of cultured, Ag-pulsed dendritic cells could significantly boost CD8 responses to whole-protein Ags in poorly cross-priming donors. Interestingly, freshly isolated monocytes performed almost as well as dendritic cells in inducing CD8 responses via cross-priming. In conclusion, the efficiency of cross-priming appears to be poor in most donors and is dependent upon properties of the individual’s APC and/or T cell repertoire. It remains unknown whether cross-priming ability translates into any clinical advantage in ability to induce CD8+ T cell responses to foreign Ags.

For immune monitoring studies during HIV vaccine clinical trials, whole blood specimens from HIV seropositive (HIV+) patients may be collected at multiple sites and sent to a central location for peripheral blood mononuclear cell (PBMC) isolation, cryopreservation and functional evaluation. In this study we show a comparison of two PBMC preparation options, Ficoll density gradient separation (Ficoll) and Cell Preparation Tubes (CPT) using shipped whole blood specimens from 19 HIV+ patients (CD4 > 350, viral load < 50). The pre- and post- cryopreservation performance of samples collected by these two methods were compared by assessment of antigen-specific IFNγ expression in CD8+ and CD8- T cells, cellular viability, and cellular recovery. The results indicate that cryopreserved PBMC samples tested for CMV- and HIV- specific interferon-gamma (IFNγ) expression performed equivalent to the respective fresh PBMC processed under both collection conditions. Compared to fresh PBMC, the viability was significantly lower for cryopreserved PBMC derived using Ficoll, although it was never less than 90%. There were no significant differences in the IFNγ response, viability, or recovery between cryopreserved PBMC derived by Ficoll and by CPT. These data suggest that CPT is an efficient system for the collection and cryopreservation of functionally active HIV+ PBMC, as well as a viable alternative to Ficoll gradient separation.

Background Cryopreservation of PBMC and/or overnight shipping of samples are required for many clinical trials, despite their potentially adverse effects upon immune monitoring assays such as MHC-peptide tetramer staining, cytokine flow cytometry (CFC), and ELISPOT. In this study, we compared the performance of these assays on leukapheresed PBMC shipped overnight in medium versus cryopreserved PBMC from matched donors. Results Using CMV pp65 peptide pool stimulation or pp65 HLA-A2 tetramer staining, there was significant correlation between shipped and cryopreserved samples for each assay (p ≤ 0.001). The differences in response magnitude between cryopreserved and shipped PBMC specimens were not significant for most antigens and assays. There was significant correlation between CFC and ELISPOT assay using pp65 peptide pool stimulation, in both shipped and cryopreserved samples (p ≤ 0.001). Strong correlation was observed between CFC (using HLA-A2-restricted pp65 peptide stimulation) and tetramer staining (p < 0.001). Roughly similar sensitivity and specificity were observed between the three assays and between shipped and cryopreserved samples for each assay. Conclusion We conclude that all three assays show concordant results on shipped versus cryopreserved specimens, when using a peptide-based readout. The assays are also concordant with each other in pair wise comparisons using equivalent antigen systems.

Background: Cytokine flow cytometry (CFC) provides a multiparameter alternative to ELISPOT assays for rapid quantitation of antigen-specific T cells. To increase the throughput of CFC assays, we have optimized methods for stimulating, staining, and acquiring whole blood or PBMC samples in 96-well or 24-well plates. Results: We have developed a protocol for whole blood stimulation and processing in deep-well 24- or 96-well plates, and fresh or cryopreserved peripheral blood mononuclear cell (PBMC) stimulation and processing in conventional 96-well round-bottom plates. Samples from both HIV1-seronegative and HIV-1-seropositive donors were tested. We show that the percent response, staining intensity, and cell recovery are comparable to stimulation and processing in tubes using traditional methods. We also show the equivalence of automated gating templates to manual gating for CFC data analysis. Conclusion: When combined with flow cytometry analysis using an automated plate loader and an automated analysis algorithm, these plate-based methods provide a higher throughput platform for CFC, as well as reducing operator-induced variability. These factors will be important for processing the numbers of samples required in large clinical trials, and for epitope mapping of patient responses.