Oligonucleotide library screening for identification of virus-specific T-cell receptors.

We demonstrate an optimized oligonucleotide library-based approach for the identification of virus-reactive T-cell receptors using Epstein-Barr virus as an example. HEK293T served as antigen-presenting cells and were co-cultured with human T cells that were transduced with T-cell receptors in question. T-cell activation was detected by CD137 expression.

Tocilizumab administration in cytokine release syndrome is associated with hypofibrinogenemia after chimeric antigen receptor T-cell therapy for hematologic malignancies.

Chimeric antigen receptor (CAR-) T cell therapy causes serious side effects including cytokine release syndrome (CRS). CRS-related coagulopathy is associated with hypofibrinogenemia that is thus far considered the result of disseminated intravascular coagulation (DIC) and liver dysfunction. We investigated incidence and risk factors for hypofibrinogenemia in 41 consecutive adult patients with hematologic malignancies (median age 69 years, range 38-83 years) receiving CAR-T cell therapy between 01/2020 and 05/2023 at the University Medical Center Regensburg. CRS occurred in 93% of patients and was accompanied by hypofibrinogenemia already from CRS grade 1. Yet, DIC and liver dysfunction mainly occurred in severe CRS (≥ grade 3). After an initial increase during CRS, fibrinogen levels dropped after administration of tocilizumab in a dose dependent manner (r = -0.44, p = 0.004). In contrast, patients who did not receive tocilizumab had increased fibrinogen levels. Logistic regression analysis identified tocilizumab as an independent risk factor for hypofibrinogenemia (odds ratio = 486, p < 0.001). We thus hypothesize that fibrinogen synthesis in CRS is upregulated in an interleukin-6-dependent acute phase reaction compensating for CRS-induced consumption of coagulation factors. Tocilizumab inhibits fibrinogen upregulation resulting in prolonged hypofibrinogenemia. These observations provide novel insights into the pathophysiology of hypofibrinogenemia following CAR-T cell therapy and emphasize the need for close fibrinogen monitoring after tocilizumab treatment of CRS.

Virus-reactive T cells expanded in aplastic anemia eliminate hematopoietic progenitor cells by molecular mimicry.

ABSTRACT: Acquired aplastic anemia is a bone marrow failure syndrome characterized by hypocellular bone marrow and peripheral blood pancytopenia. Frequent clinical responses to calcineurin inhibition and antithymocyte globulin strongly suggest critical roles for hematopoietic stem/progenitor cell-reactive T-cell clones in disease pathophysiology; however, their exact contribution and antigen specificities remain unclear. We determined differentiation states and targets of dominant T-cell clones along with their potential to eliminate hematopoietic progenitor cells in the bone marrow of 15 patients with acquired aplastic anemia. Single-cell sequencing and immunophenotyping revealed oligoclonal expansion and effector differentiation of CD8+ T-cell compartments. We reexpressed 28 dominant T-cell receptors (TCRs) of 9 patients in reporter cell lines to determine reactivity with (1) in vitro-expanded CD34+ bone marrow, (2) CD34- bone marrow, or (3) peptide pools covering immunodominant epitopes of highly prevalent viruses. Besides 5 cytomegalovirus-reactive TCRs, we identified 3 TCRs that recognized antigen presented on hematopoietic progenitor cells. T cells transduced with these TCRs eliminated hematopoietic progenitor cells of the respective patients in vitro. One progenitor cell-reactive TCR (11A5) also recognized an epitope of the Epstein-Barr virus-derived latent membrane protein 1 (LMP1) presented on HLA-A∗02:01. We identified 2 LMP1-related mimotopes within the human proteome as activating targets of TCR 11A5, providing proof of concept that molecular mimicry of viral and self-epitopes can drive T cell-mediated elimination of hematopoietic progenitor cells in aplastic anemia.

HLA-C*04:09N is expressed at the cell surface and triggers peptide-specific T-cell activation.

The null allele HLA-C*04:09N differs from HLA-C*04:01 in a frameshift mutation within its cytoplasmic domain, resulting in translation of 32 additional amino acids that are assumed to prevent cell surface expression. However, we recently identified a multiple myeloma-reactive T-cell receptor (TCR) that appeared to recognize antigen presented on HLA-C*04:09N and encouraged us to ask whether HLA-C*04:09N, albeit not easily detectable at the cell surface, can present antigen sufficient for T-cell activation. We generated two HLA-class I-deficient cell lines, re-expressed HLAC* 04:09N, detected HLA expression by flow cytometry, and tested for T-cell activation using a cytomegalovirus peptide- specific HLA-C*04:01-restricted TCR. In both cell lines, HLA-C*04:09N expression was detectable at the cell surface and could be enhanced by IFN-γ exposure. Recombinant HLA-C*04:09N expression was sufficient for T-cell activation in vitro, which could be blocked by an HLA-class I-specific antibody, suggesting HLA-TCR interaction at the cell surface. Peripheral blood mononuclear cells isolated from an individual who physiologically expressed HLA-C*04:09N triggered peptide-specific T-cell activation, confirming our results with cells with natural HLA expression levels. In conclusion, we present peptide-specific HLA-C*04:09N-restricted T-cell activation and suggest consideration of this allele in the appropriate clinical context, such as allogeneic stem cell transplantation, or in the setting of cellular therapy.

Prevention of CMV/EBV reactivation by double-specific T cells in patients after allogeneic stem cell transplantation: results from the randomized phase I/IIa MULTIVIR-01 study.

Introduction: Allogeneic stem cell transplantation is used to cure hematologic malignancies or deficiencies of the hematopoietic system. It is associated with severe immunodeficiency of the host early after transplant and therefore early reactivation of latent herpesviruses such as CMV and EBV within the first 100 days are frequent. Small studies and case series indicated that application of herpes virus specific T cells can control and prevent disease in this patient population. Methods: We report the results of a randomized controlled multi centre phase I/IIa study (MULTIVIR-01) using a newly developed T cell product with specificity for CMV and EBV derived from the allogeneic stem cell grafts used for transplantation. The study aimed at prevention and preemptive treatment of both viruses in patients after allogeneic stem cell transplantation targeting first infusion on day +30. Primary endpoints were acute transfusion reaction and acute-graft versus-host-disease after infusion of activated T cells. Results: Thirty-three patients were screened and 9 patients were treated with a total of 25 doses of the T cell product. We show that central manufacturing can be achieved successfully under study conditions and the product can be applied without major side effects. Overall survival, transplant related mortality, cumulative incidence of graft versus host disease and number of severe adverse events were not different between treatment and control groups. Expansion of CMV/EBV specific T cells was observed in a fraction of patients, but overall there was no difference in virus reactivation. Discussion: Our study results indicate peptide stimulated epitope specific T cells derived from stem cell grafts can be administered safely for prevention and preemptive treatment of reactivation without evidence for induction of acute graft versus host disease. Clinical trial registration: https://clinicaltrials.gov, identifier NCT02227641.

Patient-tailored adoptive immunotherapy with EBV-specific T cells from related and unrelated donors.

BACKGROUNDAdoptive transfer of EBV-specific T cells can restore specific immunity in immunocompromised patients with EBV-associated complications.METHODSWe provide results of a personalized T cell manufacturing program evaluating donor, patient, T cell product, and outcome data. Patient-tailored clinical-grade EBV-specific cytotoxic T lymphocyte (EBV-CTL) products from stem cell donors (SCDs), related third-party donors (TPDs), or unrelated TPDs from the allogeneic T cell donor registry (alloCELL) at Hannover Medical School were manufactured by immunomagnetic selection using a CliniMACS Plus or Prodigy device and the EBV PepTivators EBNA-1 and Select. Consecutive manufacturing processes were evaluated, and patient outcome and side effects were retrieved by retrospective chart analysis.RESULTSForty clinical-grade EBV-CTL products from SCDs, related TPDs, or unrelated TPDs were generated for 37 patients with refractory EBV infections or EBV-associated malignancies with and without a history of transplantation, within 5 days (median) after donor identification. Thirty-four patients received 1-14 EBV-CTL products (fresh and cryopreserved). EBV-CTL transfer led to a complete response in 20 of 29 patients who were evaluated for clinical response. No infusion-related toxicity was reported. EBV-specific T cells in patients' blood were detectable in 16 of 18 monitored patients (89%) after transfer, and their presence correlated with clinical response.CONCLUSIONPersonalized clinical-grade manufacture of EBV-CTL products via immunomagnetic selection from SCDs, related TPDs, or unrelated TPDs in a timely manner is feasible. Overall, EBV-CTLs were clinically effective and well tolerated. Our data suggest EBV-CTL transfer as a promising therapeutic approach for immunocompromised patients with refractory EBV-associated diseases beyond HSCT, as well as patients with preexisting organ dysfunction.TRIAL REGISTRATIONNot applicable.FUNDINGThis study was funded in part by the German Research Foundation (DFG, 158989968/SFB 900), the Deutsche Kinderkrebsstiftung (DKS 2013.09), Wilhelm-Sander-Stiftung (reference 2015.097.1), Ellen-Schmidt-Program of Hannover Medical School, and German Federal Ministry of Education and Research (reference 01EO0802).

Immune phenotypes and checkpoint molecule expression of clonally expanded lymph node-infiltrating T cells in classical Hodgkin lymphoma.

Lymph node-infiltrating T cells have been of particular interest in classical Hodgkin lymphoma (cHL). High rates of complete therapeutic responses to antibody-mediated immune checkpoint blockade, even in relapsed/refractory patients, suggest the existence of a T cell-dominated, antigen-experienced, functionally inhibited and lymphoma-directed immune microenvironment. We asked whether clonally expanded T cells (1) were detectable in cHL lymph nodes, (2) showed characteristic immune phenotypes, and (3) were inhibited by immune checkpoint molecule expression. We applied high-dimensional FACS index sorting and single cell T cell receptor αß sequencing to lymph node-infiltrating T cells from 10 treatment-naïve patients. T cells were predominantly CD4+ and showed memory differentiation. Expression of classical immune checkpoint molecules (CTLA-4, PD-1, TIM-3) was generally low (< 12.0% of T cells) and not different between CD4+ and CD8+ T cells. Degrees of clonal T cell expansion varied between patients (range: 1-18 expanded clones per patient) and was almost exclusively restricted to CD8+ T cells. Clonally expanded T cells showed non-naïve phenotypes and low checkpoint molecule expression similar to non-expanded T cells. Our data suggest that the therapeutic effects of immune checkpoint blockade require mechanisms in addition to dis-inhibition of pre-existing lymphoma-directed T cell responses. Future studies on immune checkpoint blockade-associated effects will identify molecular T cell targets, address dynamic aspects of cell compositions over time, and extend their focus beyond lymph node-infiltrating T cells.

Immune Phenotypes and Target Antigens of Clonally Expanded Bone Marrow T Cells in Treatment-Naïve Multiple Myeloma.

Multiple myeloma is a hematologic malignancy of monoclonal plasma cells that accumulate in the bone marrow. Despite their clinical and pathophysiologic relevance, the roles of bone marrow-infiltrating T cells in treatment-naïve patients are incompletely understood. We investigated whether clonally expanded T cells (i) were detectable in multiple myeloma bone marrow, (ii) showed characteristic immune phenotypes, and (iii) whether dominant clones recognized antigens selectively presented on multiple myeloma cells. Single-cell index sorting and T-cell receptor (TCR) αß sequencing of bone marrow T cells from 13 treatment-naïve patients showed dominant clonal expansion within CD8+ cytolytic effector compartments, and only a minority of expanded T-cell clones expressed the classic immune-checkpoint molecules PD-1, CTLA-4, or TIM-3. To identify their molecular targets, TCRs of 68 dominant bone marrow clones from five selected patients were reexpressed and incubated with multiple myeloma and non-multiple myeloma cells from corresponding patients. Only 1 of 68 TCRs recognized antigen presented on multiple myeloma cells. This TCR was HLA-C-restricted, self-peptide-specific and could be activated by multiple myeloma cells of multiple patients. The remaining dominant T-cell clones did not recognize multiple myeloma cells and were, in part, specific for antigens associated with chronic viral infections. In conclusion, we showed that dominant bone marrow T-cell clones in treatment-naïve patients rarely recognize antigens presented on multiple myeloma cells and exhibit low expression of classic immune-checkpoint molecules. Our data provide experimental context for experiences from clinical immune-checkpoint inhibition trials and will inform future T cell-dependent therapeutic strategies.

Rapid single-cell identification of Epstein-Barr virus-specific T-cell receptors for cellular therapy.

BACKGROUND AND AIMS: Epstein-Barr virus (EBV) is associated with solid and hematopoietic malignancies. After allogeneic stem cell transplantation, EBV infection or reactivation represents a potentially life-threatening condition with no specific treatment available in clinical routine. In vitro expansion of naturally occurring EBV-specific T cells for adoptive transfer is time-consuming and influenced by the donor's T-cell receptor (TCR) repertoire and requires a specific memory compartment that is non-existent in seronegative individuals. The authors present highly efficient identification of EBV-specific TCRs that can be expressed on human T cells and recognize EBV-infected cells. METHODS AND RESULTS: Mononuclear cells from six stem cell grafts were expanded in vitro with three HLA-B*35:01- or four HLA-A*02:01-presented peptides derived from six EBV proteins expressed during latent and lytic infection. Epitope-specific T cells expanded on average 42-fold and were single-cell-sorted and TCRαß-sequenced. To confirm specificity, 11 HLA-B*35:01- and six HLA-A*02:01-restricted dominant TCRs were expressed on reporter cell lines, and 16 of 17 TCRs recognized their presumed target peptides. To confirm recognition of virus-infected cells and assess their value for adoptive therapy, three selected HLA-B*35:01- and four HLA-A*02:01-restricted TCRs were expressed on human peripheral blood lymphocytes. All TCR-transduced cells recognized EBV-infected lymphoblastoid cell lines. CONCLUSIONS: The authors' approach provides sets of EBV epitope-specific TCRs in two different HLA contexts. Resulting cellular products do not require EBV-seropositive donors, can be adjusted to cell subsets of choice with exactly defined proportions of target-specific T cells, can be tracked in vivo and will help to overcome unmet clinical needs in the treatment and prophylaxis of EBV reactivation and associated malignancies.

Isolation of Neoantigen-Specific Human T Cell Receptors from Different Human and Murine Repertoires.

(1) Background: Mutation-specific T cell receptor (TCR)-based adoptive T cell therapy represents a truly tumor-specific immunotherapeutic strategy. However, isolating neoepitope-specific TCRs remains a challenge. (2) Methods: We investigated, side by side, different TCR repertoires-patients' peripheral lymphocytes (PBLs) and tumor-infiltrating lymphocytes (TILs), PBLs of healthy donors, and a humanized mouse model-to isolate neoepitope-specific TCRs against eight neoepitope candidates from a colon cancer and an ovarian cancer patient. Neoepitope candidates were used to stimulate T cells from different repertoires in vitro to generate neoepitope-specific T cells and isolate the specific TCRs. (3) Results: We isolated six TCRs from healthy donors, directed against four neoepitope candidates and one TCR from the murine T cell repertoire. Endogenous processing of one neoepitope, for which we isolated one TCR from both human and mouse-derived repertoires, could be shown. No neoepitope-specific TCR could be generated from the patients' own repertoire. (4) Conclusion: Our data indicate that successful isolation of neoepitope-specific TCRs depends on various factors such as the heathy donor's TCR repertoire or the presence of a tumor microenvironment allowing neoepitope-specific immune responses of the host. We show the advantage and feasibility of using healthy donor repertoires and humanized mouse TCR repertoires to generate mutation-specific TCRs with different specificities, especially in a setting when the availability of patient material is limited.

Localization-associated immune phenotypes of clonally expanded tumor-infiltrating T cells and distribution of their target antigens in rectal cancer.

The degree and type of T cell infiltration influence rectal cancer prognosis regardless of classical tumor staging. We asked whether clonal expansion and tumor infiltration are restricted to selected-phenotype T cells; which clones are accessible in peripheral blood; and what the spatial distribution of their target antigens is. From five rectal cancer patients, we isolated paired tumor-infiltrating T cells (TILs) and T cells from unaffected rectum mucosa (TUM) using 13-parameter FACS single cell index sorting. TCRαß sequences, cytokine, and transcription factor expression were determined with single cell sequencing. TILs and TUM occupied distinct phenotype compartments and clonal expansion predominantly occurred within CD8+ T cells. Expanded TIL clones identified by paired TCRαß sequencing and exclusively detectable in the tumor showed characteristic PD-1 and TIM-3 expression. TCRß repertoire sequencing identified 49 out of 149 expanded TIL clones circulating in peripheral blood and 41 (84%) of these were PD-1- TIM-3-. To determine whether clonal expansion of predominantly tumor-infiltrating T cell clones was driven by antigens uniquely presented in tumor tissue, selected TCRs were reconstructed and incubated with cells isolated from corresponding tumor or unaffected mucosa. The majority of clones exclusively detected in the tumor recognized antigen at both sites. In summary, rectal cancer is infiltrated with expanded distinct-phenotype T cell clones that either i) predominantly infiltrate the tumor, ii) predominantly infiltrate the unaffected mucosa, or iii) overlap between tumor, unaffected mucosa, and peripheral blood. However, the target antigens of predominantly tumor-infiltrating TIL clones do not appear to be restricted to tumor tissue.

FACS single cell index sorting is highly reliable and determines immune phenotypes of clonally expanded T cells.

FACS index sorting allows the isolation of single cells with retrospective identification of each single cell's high-dimensional immune phenotype. We experimentally determine the error rate of index sorting and combine the technology with T cell receptor sequencing to identify clonal T cell expansion in aplastic anemia bone marrow as an example.

Clonal Expansion and Interrelatedness of Distinct B-Lineage Compartments in Multiple Myeloma Bone Marrow.

Multiple myeloma is characterized by the clonal expansion of malignant plasma cells in the bone marrow. But the phenotypic diversity and the contribution of less predominant B-lineage clones to the biology of this disease have been controversial. Here, we asked whether cells bearing the dominant multiple myeloma immunoglobulin rearrangement occupy phenotypic compartments other than that of plasma cells. To accomplish this, we combined 13-parameter FACS index sorting and t-Stochastic Neighbor Embedding (t-SNE) visualization with high-throughput single-cell immunoglobulin sequencing to track selected B-lineage clones across different stages of human B-cell development. As expected, the predominant clones preferentially mapped to aberrant plasma cell compartments, albeit phenotypically altered from wild type. Interestingly, up to 1.2% of cells of the predominant clones colocalized with B-lineage cells of a normal phenotype. In addition, minor clones with distinct immunoglobulin sequences were detected in up to 9% of sequenced cells, but only 2 out of 12 of these clones showed aberrant immune phenotypes. The majority of these minor clones showed intraclonal silent nucleotide differences within the CDR3s and varying frequencies of somatic mutations in the immunoglobulin genes. Therefore, the phenotypic range of multiple myeloma cells in the bone marrow is not confined to aberrant-phenotype plasma cells but extends to low frequencies of normal-phenotype B cells, in line with the recently reported success of B cell-targeting cellular therapies in some patients. The majority of minor clones result from parallel nonmalignant expansion. Cancer Immunol Res; 5(9); 744-54. ©2017 AACR.

[Flow-cytometric immunophenotyping in clinical diagnostics]. / Durchflusszytometrische Immunphänotypisierung in der klinischen Diagnostik.

Flow cytometric immunophenotyping represents an indispensable tool in hematological and immunological diagnostics. The most frequent indications include lymphocyte phenotyping and the diagnosis and monitoring of benign and malignant hematologic diseases. The role of immunophenotyping in clinical practice is evolving rapidly. This review provides an overview of its current applications and limitations.

Single-cell systems-level analysis of human Toll-like receptor activation defines a chemokine signature in patients with systemic lupus erythematosus.

BACKGROUND: Activation of Toll-like receptors (TLRs) induces inflammatory responses involved in immunity to pathogens and autoimmune pathogenesis, such as in patients with systemic lupus erythematosus (SLE). Although TLRs are differentially expressed across the immune system, a comprehensive analysis of how multiple immune cell subsets respond in a system-wide manner has not been described. OBJECTIVE: We sought to characterize TLR activation across multiple immune cell subsets and subjects, with the goal of establishing a reference framework against which to compare pathologic processes. METHODS: Peripheral whole-blood samples were stimulated with TLR ligands and analyzed by means of mass cytometry simultaneously for surface marker expression, activation states of intracellular signaling proteins, and cytokine production. We developed a novel data visualization tool to provide an integrated view of TLR signaling networks with single-cell resolution. We studied 17 healthy volunteer donors and 8 patients with newly diagnosed and untreated SLE. RESULTS: Our data revealed the diversity of TLR-induced responses within cell types, with TLR ligand specificity. Subsets of natural killer cells and T cells selectively induced nuclear factor κ light chain enhancer of activated B cells in response to TLR2 ligands. CD14(hi) monocytes exhibited the most polyfunctional cytokine expression patterns, with more than 80 distinct cytokine combinations. Monocytic TLR-induced cytokine patterns were shared among a group of healthy donors, with minimal intraindividual and interindividual variability. Furthermore, autoimmune disease altered baseline cytokine production; newly diagnosed untreated SLE patients shared a distinct monocytic chemokine signature, despite clinical heterogeneity. CONCLUSION: Mass cytometry defined a systems-level reference framework for human TLR activation, which can be applied to study perturbations in patients with inflammatory diseases, such as SLE.

Mass cytometry analysis shows that a novel memory phenotype B cell is expanded in multiple myeloma.

It would be very beneficial if the status of cancers could be determined from a blood specimen. However, peripheral blood leukocytes are very heterogeneous between individuals, and thus high-resolution technologies are likely required. We used cytometry by time-of-flight and next-generation sequencing to ask whether a plasma cell cancer (multiple myeloma) and related precancerous states had any consistent effect on the peripheral blood mononuclear cell phenotypes of patients. Analysis of peripheral blood samples from 13 cancer patients, 9 precancer patients, and 9 healthy individuals revealed significant differences in the frequencies of the T-cell, B-cell, and natural killer-cell compartments. Most strikingly, we identified a novel B-cell population that normally accounts for 4.0% ± 0.7% (mean ± SD) of total B cells and is up to 13-fold expanded in multiple myeloma patients with active disease. This population expressed markers previously associated with both memory (CD27(+)) and naïve (CD24(lo)CD38(+)) phenotypes. Single-cell immunoglobulin gene sequencing showed polyclonality, indicating that these cells are not precursors to the myeloma, and somatic mutations, a characteristic of memory cells. SYK, ERK, and p38 phosphorylation responses, and the fact that most of these cells expressed isotypes other than IgM or IgD, confirmed the memory character of this population, defining it as a novel type of memory B cells.

Linking T-cell receptor sequence to functional phenotype at the single-cell level.

Although each T lymphocyte expresses a T-cell receptor (TCR) that recognizes cognate antigen and controls T-cell activation, different T cells bearing the same TCR can be functionally distinct. Each TCR is a heterodimer, and both α- and ß-chains contribute to determining TCR antigen specificity. Here we present a methodology enabling integration of information about TCR specificity with information about T cell function. This method involves sequencing of TCRα and TCRß genes, and amplifying functional genes characteristic of different T cell subsets, in single T cells. Because this approach retains information about individual TCRα-TCRß pairs, TCRs of interest can be expressed and used in functional studies, for antigen discovery, or in therapeutic applications. We apply this approach to study the clonal ancestry and differentiation of T lymphocytes infiltrating a human colorectal carcinoma.

The enhancer and promoter landscape of human regulatory and conventional T-cell subpopulations.

CD4(+)CD25(+)FOXP3(+) human regulatory T cells (Tregs) are essential for self-tolerance and immune homeostasis. Here, we describe the promoterome of CD4(+)CD25(high)CD45RA(+) naïve and CD4(+)CD25(high)CD45RA(-) memory Tregs and their CD25(-) conventional T-cell (Tconv) counterparts both before and after in vitro expansion by cap analysis of gene expression (CAGE) adapted to single-molecule sequencing (HeliScopeCAGE). We performed comprehensive comparative digital gene expression analyses and revealed novel transcription start sites, of which several were validated as alternative promoters of known genes. For all in vitro expanded subsets, we additionally generated global maps of poised and active enhancer elements marked by histone H3 lysine 4 monomethylation and histone H3 lysine 27 acetylation, describe their cell type-specific motif signatures, and evaluate the role of candidate transcription factors STAT5, FOXP3, RUNX1, and ETS1 in both Treg- and Tconv-specific enhancer architectures. Network analyses of gene expression data revealed additional candidate transcription factors contributing to cell type specificity and a transcription factor network in Tregs that is dominated by FOXP3 interaction partners and targets. In summary, we provide a comprehensive and easily accessible resource of gene expression and gene regulation in human Treg and Tconv subpopulations.

Effects of serum and plasma matrices on multiplex immunoassays.

Multiplexed fluorescence or electrochemiluminescence immunoassays of soluble cytokines are commonly performed in the context of human serum or plasma, to look for disease biomarkers and to monitor the immune system in a simple and minimally invasive way. These assays provide challenges due to the complexities of the matrix (serum or plasma) and the presence of many cytokines near the limit of detection of the assay. Here, we compare the readout of matched serum and plasma samples, which are generally correlated. However, a subset of cytokines usually have higher levels in serum, and the non-specific background is significantly increased in serum versus plasma. Presumably as a result of this non-specific background, disease-related decreases in low-abundance cytokines can sometimes be detected in plasma but not in serum. We further show, through spike recovery experiments, that both serum and plasma inhibit the readout of many cytokines, with some variability between donors, but with serum causing greater inhibition than plasma in many cases. Standard diluents from different vendors can partially reverse this inhibition to varying degrees. Dilution of samples can also partly overcome the inhibitory effect of the matrix. We also show that dilution is nonlinear and differentially affects various cytokines. Together, these data argue that (1) plasma is a more sensitive matrix for detecting changes in certain low-abundance cytokines; (2) calculation of concentrations in serum or plasma matrices is inherently inaccurate; and (3) dilution of samples should not be assumed to be linear, i.e., all comparisons need to be made among similarly diluted samples.