ABSTRACT Background Little is known about the ontogeny of T cell immunity during infancy in farming and urban lifestyles due to the lack of immunophenotyping in such birth cohorts. Methods Two birth cohorts (farming and urban) at differing risks and rates of allergic diseases were compared. Blood mononuclear cells were collected from infants at birth, and 6 and 12 months of age. Full spectrum flow cytometry, followed by traditional gating and the Scalable Weighted Iterative Flow‐clustering Technique (SWIFT) high‐dimensional analysis, were used to identify cell populations that differed between farming and urban infants. Additionally, single‐cell RNAseq and multiplex cytokine assays were used to assess the function of cell populations of interest. Results Several regulatory T cell (Treg) subpopulations were elevated in farming lifestyles and in non‐atopic infants. A unique effector memory CD25 + CD127 + CD161 − CD49d + CCR4 + CRTH2 + Th2 population was elevated at 6 months in urban infants and in those who developed atopic dermatitis and/or food allergy and allergic sensitization. Although this population shared Th2 and IL‐9 skewing with Th2A cells, the population uniquely failed to express CD161, produced more IL‐2 and TNF‐α, and upregulated the differentially expressed genes (DEGs), FOXP3 and the cytokine inducible SH2‐containing protein gene ( CISH ) relative to Th2A cells. This population has been termed Th2B cells. Conclusion We describe a unique effector memory Th2 population elevated in urban high‐risk infants, potentially implicated in the development of allergic disease.

Multi-drug combination strategy targeting three different molecules involved in different pathways to overcome single-agent resistance in relapsed/refractory CLL patients. The clinical trial utilized 1)a therapeutic anti-CD20 monoclonal antibody (mAb), ublituximab, which destroys CLL cells by an antibody-dependent cellular phagocytosis (ADCP) mechanism; 2)a B cell receptor (BCR) signaling inhibitor, umbralisib, which blocks PI3Kẟ; 3)and an anti-apoptosis inhibitor, venetoclax, which blocks cell survival promoted by BCL-2 that stops mitochondria from initiating apoptosis. Our correlative study focused on the first two treatments prior to venetoclax addition. We found that patients respond to anti-CD20 antibody and BCR signaling inhibition with rapid reductions in CLL cell counts and CD20 levels. Standard high dose (375 mg/m2) anti-CD20 antibody treatment significantly decreased CLL surface CD20 levels, potentially limiting treatment efficacy. Anti-CD20 antibody plus B cell receptor signaling inhibition reduced CLL cell counts and lymph node tumors, enabling BCL-2 inhibitor treatment to avoid tumor lysis syndrome.

Vaccination remains our main defence against influenza, which causes substantial annual mortality and poses a serious pandemic threat. Influenza virus evades immunity by rapidly changing its surface antigens but, even when the vaccine is well matched to the current circulating virus strains, influenza vaccines are not as effective as many other vaccines. Influenza vaccine development has traditionally focused on the induction of protective antibodies, but there is mounting evidence that T cell responses are also protective against influenza. Thus, future vaccines designed to promote both broad T cell effector functions and antibodies may provide enhanced protection. As we discuss, such vaccines present several challenges that require new strategic and economic considerations. Vaccine-induced T cells relevant to protection may reside in the lungs or lymphoid tissues, requiring more invasive assays to assess the immunogenicity of vaccine candidates. T cell functions may contain and resolve infection rather than completely prevent infection and early illness, requiring vaccine effectiveness to be assessed based on the prevention of severe disease and death rather than symptomatic infection. It can be complex and costly to measure T cell responses and infrequent clinical outcomes, and thus innovations in clinical trial design are needed for economic reasons. Nevertheless, the goal of more effective influenza vaccines justifies renewed and intensive efforts. Compared with many other vaccines, current vaccines against influenza provide only limited protection. Here, the authors describe the challenges and recent attempts at generating T cell-based vaccines. It may be important to combine T cell-based vaccines with antibody-based vaccines to provide long-lasting immunity across influenza virus strains.

IntroductionThe HVTN 105 vaccine clinical trial tested four combinations of two immunogens - the DNA vaccine DNA-HIV-PT123, and the protein vaccine AIDSVAX B/E. All combinations induced substantial antibody and CD4+ T cell responses in many participants. We have now re-examined the intracellular cytokine staining flow cytometry data using the high-resolution SWIFT clustering algorithm, which is very effective for enumerating rare populations such as antigen-responsive T cells, and also determined correlations between the antibody and T cell responses.MethodsFlow cytometry samples across all the analysis batches were registered using the swiftReg registration tool, which reduces batch variation without compromising biological variation. Registered data were clustered using the SWIFT algorithm, and cluster template competition was used to identify clusters of antigen-responsive T cells and to separate these from constitutive cytokine producing cell clusters.ResultsRegistration strongly reduced batch variation among batches analyzed across several months. This in-depth clustering analysis identified a greater proportion of responders than the original analysis. A subset of antigen-responsive clusters producing IL-21 was identified. The cytokine patterns in each vaccine group were related to the type of vaccine – protein antigens tended to induce more cells producing IL-2 but not IFN-γ, whereas DNA vaccines tended to induce more IL-2+ IFN-γ+ CD4 T cells. Several significant correlations were identified between specific antibody responses and antigen-responsive T cell clusters. The best correlations were not necessarily observed with the strongest antibody or T cell responses.ConclusionIn the complex HVTN105 dataset, alternative analysis methods increased sensitivity of the detection of antigen-specific T cells; increased the number of identified vaccine responders; identified a small IL-21-producing T cell population; and demonstrated significant correlations between specific T cell populations and serum antibody responses. Multiple analysis strategies may be valuable for extracting the most information from large, complex studies.

Little is known about T effector cells in early allergic disease development due to lack of immunophenotyping in birth cohorts. We sought to further examine these T cell populations in early infancy. Our study includes two birth cohorts (farming and urban) at differing risks and rates of allergic diseases. In this study, blood mononuclear cells were collected from infants at birth, and 6 and 12 months of age. We used full spectrum flow cytometry followed by traditional gating and the Scalable Weighted Iterative Flow-clustering Technique (SWIFT) high dimensional analysis to identify cell populations that differed between farming and urban infants. Additionally, we utilized RNAseq and Luminex to assess the function of the identified cell population. Through SWIFT, we identified a novel effector memory CD25+CD127+ Th2-like population that is elevated at 6 months in urban infants at high risk for allergic disease as well as infants who developed atopic dermatitis and/or food allergy and allergic sensitization. Functional assays confirmed this population to be Th2-skewed, as evidenced by an increase in Th2 cytokines and upregulation of pathways linked to asthma and Th2 differentiation. We also identified a TIGIT+ regulatory T cell (Treg) subpopulation that was elevated in farming lifestyle as well as in non-atopic infants. Overall, we have discovered a previously unidentified Th2 subset implicated in the development of allergic disease as well as a Treg subpopulation associated with protection against allergic disease. These novel findings will advance research in the mechanisms of development and prevention of allergic diseases in childhood.

Background: Atopic dermatitis (AD) is an inflammatory disorder characterized by dominant type 2 inflammation leading to chronic pruritic skin lesions, allergic comorbidities, and Staphylococcus aureus skin colonization and infections. S aureus is thought to play a role in AD severity. Objectives: This study characterized the changes in the host -microbial interface in subjects with AD following type 2 blockade with dupilumab. Methods: Participants (n 5 71) with moderate-severe AD were enrolled in a randomized (dupilumab vs placebo; 2:1), double-blind study at Atopic Dermatitis Research Network centers. Bioassays were performed at multiple time points: S aureus and virulence factor quantification, 16s ribosomal RNA microbiome, serum biomarkers, skin transcriptomic analyses, and peripheral blood T-cell phenotyping. Results: At baseline, 100% of participants were S aureus colonized on the skin surface. Dupilumab treatment resulted in significant reductions in S aureus after only 3 days (compared to placebo), which was 11 days before clinical improvement. Participants with the greatest S aureus reductions had the best clinical outcomes, and these reductions correlated with reductions in serum CCL17 and disease severity. Reductions (10-fold) in S aureus cytotoxins (day 7), perturbations in TH17-cell subsets (day 14), and increased expression of genes relevant for IL-17, neutrophil, and complement pathways (day 7) were also observed. Conclusions: Blockade of IL-4 and IL-13 signaling, very rapidly (day 3) reduces S aureus abundance in subjects with AD, and this reduction correlates with reductions in the type 2 biomarker, CCL17, and measures of AD severity (excluding itch). Immunoprofiling and/or transcriptomics suggest a role for TH17 cells, neutrophils, and complement activation as potential mechanisms to explain these findings. (J Allergy Clin Immunol 2023;152:1179-95.)

Atopic dermatitis (AD) is characterized by type 2 inflammation, chronic pruritus and Staphylococcus aureus (SA) skin colonization and infections.SA is thought to play a role in AD severity.We characterized the changes in the host-microbial interface in AD subjects following type 2 blockade. Participants (N=71) with moderate-severe AD were enrolled in a RDBPC study (dupilumab vs placebo; 2:1) at Atopic Dermatitis Research Network centers. Bioassays were performed at multiple timepoints: SA and virulence factor quantification, 16s rRNA microbiome, serum biomarkers, barrier function, skin transcriptomic analyses and PBMC phenotyping. Dupilumab treatment resulted in significant SA reductions after only 3 days; 11 days before clinical improvement. Those with the greatest SA reductions had the best clinical outcomes, and reductions correlated with CCL17 reductions. Reductions in SA cytotoxins (day 7), increases in skin-homing Th17 subsets (day 14), and increased expression of genes relevant for IL-17, neutrophil and complement pathways (day 7) were also observed. Dupilumab very rapidly reduced SA abundance which correlated with reductions in the type 2 biomarker, CCL17. Immunoprofiling and transcriptomics suggest a role for Th17 immunity, while transcriptomic analysis also points to a role for neutrophil and complement activation as potential mechanisms to explain these findings.

T cell dysregulation has been observed in many tumors, but has not been well-characterized in rare B-cell splenic lymphomas. We began defining the T cell populations in the tumor microenvironments of four human indolent B cell splenic lymphomas: splenic marginal zone lymphoma (SMZL), hairy cell leukemia (HCL), hairy cell leukemia variant (HCLv), and splenic diffuse red pulp small B-cell lymphoma (SDRPL). After Institutional Research Subjects Review Board approval, we studied de-identified cryopreserved bulk splenic tumor suspension cell isolates from fresh (less than 4 h) splenectomy tissue and matched intact formalin-fixed paraffin embedded (FFPE) tissue sections from 17 splenic lymphoma patients and 4 trauma patients (controls). Initial immunohistochemical staining analysis of FFPE tissue sections suggests that the distribution of intratumoral T cells is markedly different among B cell splenic lymphomas. In HCL, residual T cell zones appear retained, albeit diminished, while HCLv and SDRPL have markedly reduced T cells throughout with non-distinct zonation, and SMZL has prominent peritumoral collections of T cells at the interface of the neoplastic white pulp. High-parameter (32-color) fluorescence spectral flow cytometry (Cytek Aurora) analysis suggests that splenic lymphomas exhibit CD4+ and CD8+ T cells with higher proportions of transitional memory, regulatory, and exhausted phenotypes than controls. T cell activation may be enhanced in HCLv. HCLv and SMZL may have decreased naïve T cells. Finally, splenic lymphomas had higher proportions of T cells with an exhausted phenotype compared to trauma samples. In summary, our data suggests T cells are dysregulated in B-cell splenic lymphomas. American Association of Immunologists, Hairy Cell Leukemia Foundation / Sass Foundation for Medical Research, and generous donations by Elizabeth Aaron.

Intravenous (IV) anti-CD20 monoclonal antibody (mAb) therapy for chronic lymphocytic leukemia (CLL) patients often produces a first dose infusion reaction (FDIR) within the first 2 h that requires careful monitoring to avoid serious complications and fatalities. To better understand FDIR, we studied blood samples collected from 37 treatment-naïve CLL patients in our clinical trial NCT03788291 undergoing IV treatment with 50 mg of anti-CD20 mAb (rituximab). We analyzed four time points: baseline (prior to infusion), 1 h later (during infusion), at the end of infusion (~2.5 h) and at 48 h. Infusion reactions (CTCAE (v5) grade ≥2 events) were managed by interrupting the infusion and with symptomatic treatment. 24 (65%) patients had a FDIR and all patients completed the infusion. Patient and CLL characteristics including measures of disease severity (Rai stage, IGHV mutation status, cytogenetic defects) did not correlate with FDIR. High levels of CLL cells and/or CD20 and their subsequent depletion resulting in decreased serum mAb and/or complement are thought to be risks for FDIR. However, measurements of these parameters did not correlate. Induction of cytokine release syndrome (CRS) may correlate with FDIR. However, CRS was observed in all samples after mAb infusion. The only significant associations with FDIR were higher levels of IP-10, IL-6, and IL-8, with IP-10 most significant. Anti-CD20 mAb activated cytotoxic effector cells, such as tissue-resident macrophages that ingest CLL cells via antibody-dependent cellular phagocytosis (ADCP), may be responsible for these events leading to FDIR. A mouse model of anti-CD20 mAb FDIR and in vitro ADCP cell systems will be useful to further understand the biology of FDIR. Supported by funding from Acerta/AstraZeneca, the Cadregari Foundation, a generous donation by Elizabeth Aaron, and the NIH NCI grant number R21CA267040.

Monoclonal antibody induced infusion reactions (IRs) can be serious and even fatal. We used clinical data and blood samples from 37 treatment naive patients with chronic lymphocytic leukemia/small lymphocytic lym-phoma (CLL) initiating therapy for progressive disease with a single 50 mg dose of intravenous (IV) rituximab at 25 mg/h. Twenty-four (65 %) patients had IRs at a median of 78 min (range 35-128) and rituximab dose of 32 mg (range 15-50). IR risk did not correlate with patient or CLL characteristics, CLL counts or CD20 levels, or serum rituximab or complement concentrations. Thirty-five (95 %) patients had cytokine release response with a >= 4-fold increase in serum concentration of >= 1 inflammatory cytokine. IRs were associated with significantly higher post-infusion serum concentrations of gamma interferon induced cytokines IP-10, IL-6 and IL-8. IP-10 concentrations increased >= 4-fold in all patients with an IR and were above the upper limit of detection (40,000 pg/ml) in 17 (71 %). In contrast, to only three (23 %) patients without an IR had an >= 4-fold increase in serum concentrations of IP-10 (highest 22,013 pg/ml). Our data suggest that cytokine release could be initiated by activation of effector cells responsible for clearance of circulating CLL cells with IRs occurring in those with higher levels of gamma interferon induced cytokines. These novel insights could inform future research to better understand and manage IRs and understand the role of cytokines in the control of cytotoxic immune responses to mAb.

Introduction Splenic B-cell lymphomas, (SLs, total incidence ~1:100,000/year) comprise four distinct indolent B-cell non-Hodgkin lymphomas. Three predominantly involve the red pulp of the spleen (classical hairy cell leukemia (HCL), hairy cell leukemia variant (HCLv) and splenic diffuse red pulp small B-cell lymphoma (SDRPL)) and the fourth predominantly involves the white pulp (splenic marginal zone lymphoma (SMZL)). HCL is usually diagnosed by detection of an activation mutation in BRAF and responds well to treatment. We focused our study on the remaining three diseases, which have overlapping clinical presentations with only subtle differences in their morphology and phenotypes, but have considerable differences in prognosis, response to treatment and outcome. The white pulp lymphoma SMZL generally has good treatment responses and longer survival, whereas the red pulp SLs, HCLv and SDRPL are less responsive to treatments with shorter survival. Cells of the tumor microenvironment (TME) may have distinct characteristics between these three diseases. We hypothesized that TME innate immune cells from red pulp SLs (HCLv, and SDRPL) would be similar to each other and distinct from the white pulp SL, SMZL.In this study, we report that natural killer (NK) cells, which are important innate immune cells critical for immune surveillance, vary in proportions among red and white pulp SLs. Methods We studied splenectomy specimens from patients with SMZL (n=8), HCLv (n=8), SDRPL (n=7), and controls (healthy, post-trauma, n=8). Cryo-preserved bulk splenic suspension cell isolates from fresh (<4 hour) surgical specimens and intact formalin-fixed paraffin embedded (FFPE) tissue sections were used. Human specimen collection and usage was conducted with written informed consent after approval of the Institutional Research Subjects Review Board. FFPE tissue sections were studied by immunohistochemical (IHC) staining and splenic suspension cells were analyzed by high-parameter fluorescence flow cytometry using a Cytek Aurora spectral flow cytometer with a 25-color myeloid and a 28-color NK/B-cell panel. Results IHC characterization showed an extensive network of innate immune cells as reflected by CD163 + macrophages. Using high-parameter flow cytometry, we focused on innate immune cells in the non-B- non-T-cell (CD3 -CD19 -) population. The average proportions of this population were: NK cells 25-36%, monocyte/macrophages 20-34%, dendritic cells 5-11%, and granulocytes 2%. Among the patient groups, the median percentage of NK cells was greater for controls (34%) and SMZL (35%) as compared to red pulp lymphomas (HCLv (28%), SDRPL (24%)). Next, we examined NK subtypes: 1) cytotoxic NK cells (CD56 +CD16 +), 2) cytokine-secreting weakly cytotoxic NK cells (CD56 +CD16 -), and 3) undefined NK cells (CD56 -CD16 +) as a median percentage of total NK cells. The major subtype for control and SMZL was CD56 +CD16 - with values of 58 and 44%, respectively. This subtype comprises only 38%-40% of red pulp SLs. Proportionally the CD56 +CD16 + subtype was the largest for red pulp SLs at 52-57% as compared to 30% for control and 38% for SMZL. The remaining CD56 -CD16 + NK cell subtype was generally higher among red pulp SLs (HCLv (9%), SDRPL (12%)) as compared to control (6%) and SMZL (4%) patients. The increase in cytotoxic CD56 +CD16 + subtype among red pulp SLs was confirmed by measuring the percentage of granzymeB +perforin + cells among mature cytotoxic NK cells (CD56 +CD16 +/ CD57 +NKG2a -). SLs had higher proportions of this NK phenotype (78-86% compared to control 69%) and red pulp SLs had the most granzymeB +perforin + proportions with 82 and 86% for HCLv and SDRPL, respectively. Discussion In summary, spleens from patients with SMZL had similar percentages of total NK cells to controls, with lower percentages in red pulp SLs. All SLs had a higher percentage of cytotoxic NK cells than control, with the red pulp SLs having the most. These data suggest that monoclonal antibody treatments targeting SL antigen, such as anti-CD20, may be effective by activating cytotoxic NK cell to target lymphoma cells by antibody-dependent cellular cytotoxicity. Thus, uncovering the characteristics of the TME may aid the development of non-invasive diagnostic procedures and/or precision therapies.

Background: Little is known about the ontogeny of T cell immunity during infancy in farming and urban lifestyle due to lack of immunophenotyping in such birth cohorts. Methods: Our study includes two birth cohorts (farming and urban) at differing risks and rates of allergic diseases. In this study, blood mononuclear cells were collected from infants at birth, and 6 and 12 months of age. We used full spectrum flow cytometry followed by traditional gating and the Scalable Weighted Iterative Flow-clustering Technique (SWIFT) high dimensional analysis to identify cell populations that differed between farming and urban infants. Additionally, we utilized RNAseq and Luminex to assess the function of the cell population of interest. Results: We identified several regulatory T cell (Treg) subpopulations elevated in farming lifestyle as well as in non-atopic infants. We also found a unique, recently activated effector memory CD25 CD127 CD161 CCR4 CRTH2 Th2 population that is elevated at 6 months in urban infants as well as infants who developed atopic dermatitis and/or food allergy and allergic sensitization. Functional assays confirmed this population to be highly Th2-skewed, as evidenced by an increase in Th2 cytokines and upregulation of pathways linked to asthma and Th2 differentiation. Conclusion: We have discovered Treg subpopulations associated with farming lifestyle and protection against allergic disease. We also describe a unique, recently activated effector memory Th2 population elevated in urban high-risk infants that is similar to the pathogenic effector Th2A cells but is CD25 and CD161 , potentially representing pre-Th2A cells implicated in the development of allergic disease.

Indolent splenic B-cell lymphomas are rare diseases (incidence <1:100,000) that include splenic marginal zone lymphoma (SMZL), hairy cell leukemia (cHCL), hairy cell leukaemia variant (HCLv), and splenic diffuse red pulp small B-cell lymphoma (SDRPL). These splenic lymphomas can have overlapping clinical presentations with only subtle differences in their morphology and phenotypes. However, there are considerable differences in prognosis, response to treatment, and outcomes. Better understanding of the tumor microenvironment (TME) is essential to design expedient and accurate diagnostic methods and precision drug treatments for each type of splenic B-cell lymphoma. We hypothesized each splenic lymphoma would have a distinct TME with some overlap for the red pulp lymphomas (cHCL, HCLv, and SDRPL). Human specimen collection and usage was approved by the Institutional Research Subjects Review Board. We studied cryo-preserved bulk splenic tumor suspension cell isolates from fresh (<4 hour) splenectomy tissue and intact formalin-fixed paraffin embedded (FFPE) tissue sections from 17 splenic lymphoma patients and 4 trauma patients (controls). FFPE tissue sections were studied by immunohistochemical (IHC) staining and splenic suspension cells analyzed by high-parameter fluorescence flow cytometry with a 29-color myeloid panel using a Cytek Aurora spectral flow cytometer. Initial IHC characterization of the splenic TME illustrated a markedly different distribution of intratumoral T cells (CD3) and macrophages (Mθ) (CD163). In cHCL, residual T cell zones appear retained, albeit diminished, while HCLv and SDRPL have markedly reduced T cells throughout with non-distinct zonation, and SMZL has prominent peritumoral collections of T cells at the interface of the neoplastic white pulp. Tissue architecture, as evidenced by splenic macrophage distribution, was markedly different. cHCL and HCLv showed diffuse but distorted networks of interdigitating macrophages, while SDRPL retained a nearly normal sinusoidal distribution of abundant macrophages, and SMZL showed total exclusion of CD163-positive Mθ from areas of confluent neoplastic white pulp. Analysis of splenic suspension cells showed an average of 72% B cells in the lymphoma specimens, twice that of trauma spleens. T cells made up the greatest fraction of non-B cells (~63%) for both the diseased and control samples. We analyzed the non-T and non-B cells to examine the innate immune cells. Granulocytes comprised on average 4% with no difference between the splenic lymphomas or trauma (Fig.1A). Trauma spleen had 86% macrophage/monocytes (M/Mθ)s, with similar values for HCLv (80%) and cHCL (74%). The percentage of M/Mθ was lower in SDRPL (55%) and SMZL (48%). We then analyzed the percentage of M/Mθ subtypes. As shown in (Fig.1B), CD14+CD16- was the major subtype for trauma (58%) and HCLv (50%). Conversely for cHCL, CD14loCD16+ was predominant (60%) subtype. SDRPL and SMZL shared similar subtype profiles with equal proportions of CD14+CD16- and CD14loCD16+ (40%). We then measured the dendritic cells of the innate immune population. SDRPL (41%) and SMZL (48%) had higher percentages than trauma (12%), HCLv (16%), or cHCL (20%). Conventional dendritic cell 2 (cDC2) was the predominant subtype comprising 50-74% of the DCs. Average conventional dendritic cell 1 (cDC1) and plasmacytoid dendritic cells (pDCs) were 30% and 9%, respectively. pDC was lower in cHCL than the other lymphomas (1%). Our data show a similar M/Mθ component of the innate immune TME in the red pulp lymphoma SDRPL and white pulp lymphoma SMZL. SDRPL and SMZL also had higher proportions of dendritic cells compared to cHCL, HCLv, or trauma spleens. cDC2 was the predominant DC type in the spleen regardless of disease state. We are currently analyzing flow cytometry panels to access natural killer cell distribution and T cell functionality. We aim to better understand immune cell interactions in the TME and discover variations between the splenic B-cell lymphomas, resulting in improved diagnosis and therapy. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Most patients initiating anti-CD20 monoclonal antibody (mAb) therapy for CLL have a disruptive infusion reaction that can cause serious complications and be fatal. A better understanding of the risk factors and etiology of infusion reactions is required to improve preventative measures and management. We report a study to test the hypothesis that IV rituximab induces an inflammatory cytokines response with differences in individual cytokine concentrations correlating with infusion reactions. We designed a clinical trial (NCT03788291) for initial treatment of patients with progressive CLL that provided clinical data and blood specimens from 37 patients treated with 50 mg of IV rituximab infused at 25 mg/h. Blood specimens were collected immediately prior to initiation of rituximab (baseline), one hour later (1h), at the end of the infusion (post-infusion) and at 48 h after initiation of therapy (48h). Infusion reactions (CTCAE grade ≥2 events) were managed by interrupting the infusion and initiating symptomatic treatment. We measured circulating CLL cell counts and CD20 levels, and serum rituximab concentration, complement levels, and cytokine concentrations of IFN-γ, IL-10, IL-18, IL-2, IL-4, IL-6, IL-8, IP-10, MCP-1, MIP-1α, MIP-1β, and TNF-α using standard methods. Twenty-four (65%) patients had 25 infusion reactions (all CTCAE grade 2) at a median of 78 minutes (range 35-128) after initiation of rituximab therapy (median dose 32 mg, range 15-50). All patients completed the 50 mg rituximab infusion (median time 150 minutes, range 119-195). Patient and CLL characteristics including baseline CLL cell counts did not predict the risk of infusion reactions. Median CLL counts at 1h decreased to 14.7% of baseline with no further changes post-infusion and no significant difference between patients with or without infusion reactions (60x109/L vs. 45x109/L, p=0.61). Median CLL cell membrane CD20 levels (x103 molecules/cell) decreased from 4.77 at baseline to 3.10 at 1h (p=0.002) and 1.59 post-infusion (p<0.0001 vs. baseline and 1h) with no significant differences between patients with or without an infusion reaction. The median estimated number of circulating CLL cell membrane CD20 molecules (x1012 molecules/L) decreased from 325.8 at baseline to 34.1 at 1h (p<0.0001) and 14.5 post-infusion (p<0.0001 vs. baseline, p<0.03 vs. 1h) with no significant differences between patients with or without an infusion reaction. There was no correlation between risk of infusion reaction and serum levels of rituximab or serum complement. Thirty-five (95%) patients had a ≥4 fold increase in serum concentration of ≥1 cytokine with resolution of the cytokine response at 48h. All patients with an infusion reaction had significantly higher median post-infusion serum concentrations of IP-10, IL-6 and IL-8. All 17 patients with IP-10 serum concentrations above the limit of detection (40,000 pg/ml) post-infusion had an infusion reaction, and all 24 patients with an infusion reaction had a ≥4 fold increase in serum IP-10 concentrations. Of 13 patients without an infusion reaction, the highest measured serum IP-10 concentration was 22,013 pg/ml and only 3 (23%) had ≥4 fold increases in serum IP-10 concentrations. Slow infusion of a reduced dose of rituximab induced a cytokine response in 95% of patients and therapy disrupting infusion reactions occurred at a comparable rate (65%) to standard rituximab therapy. Infusion reactions were associated with increased levels of IP-10, IL-6 and IL-8. IP-10 levels were increased by ≥4 fold in all patients with an infusion reaction compared to 23% without an infusion reaction. These data suggest that rituximab infusion reactions are caused by inflammatory cytokines produced by cytotoxic effector cells activated by rituximab independent of the pre-treatment amount of circulating CLL cells or CD20 levels, or efficacy of rituximab in reducing the circulating CLL cell count. Infusion reactions occurred after at least 35 minutes of therapy concurrent with a rapid decrease in circulating CLL cells. This observation suggests that cytokine release could be a result of activation of effector cells responsible for CLL cell elimination. Our data provides novel insights into the potential mechanism of infusion reactions and could form the basis of further research to determine methods to control this clinically important and therapy limiting complication.

Figure 8 plot data for the publication: swiftReg cluster registration automatically reduces flow cytometry data variability including batch effects.