Abstract
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.
Respiratory syncytial virus (RSV) and influenza virus cause severe disease in elderly patients. The balance of pro- and anti-inflammatory cytokines may be critical in determining disease pathogenesis ...and outcome of infection. The frequencies of CD4 IL-10 (anti-inflammatory)- and CD4 and CD8 IFNγ (pro-inflammatory)-secreting memory T cells specific for either RSV or influenza were not significantly different between young and elderly groups, although the ratio of IL-10/IFNγ was significantly reduced in the elderly RSV response. A similar trend was seen for influenza. IFNγ-secreting CD4 T cells contributed significantly more to anti-RSV than anti-influenza responses in both groups.
Cytotoxicity is a key effector function of CD8 T cells. However, what proportion of antigen-specific CD8 T cells in vivo exert cytotoxic activity during a functional CD8 T-cell response to infection ...still remains unknown. We used the Lysispot assay to directly enumerate cytotoxic CD8 T cells from the spleen ex vivo during the immune response to infection with the intracellular bacterium Listeria monocytogenes. We demonstrate that not all antigen-responsive gamma interferon (IFN-γ)-secreting T cells display cytotoxic activity. Most CD8 T cells detected at early time points of the response were cytotoxic. This percentage continuously declined during both the expansion and contraction phases to about 50% at the peak and to <10% of IFN-γ-producing cells in the memory phase. As described for clonal expansion, this elaboration of a program of differentiation after an initial stimulus was not affected by antigen or CD4 help but, like proliferation, could be influenced by later reinfection. These data indicate that cytotoxic effector function during the response to infection is regulated independently from IFN-γ secretion or expansion or contraction of the overall CD8 T-cell response.
Mouse and human CD4 T cells primed during an immune response may differentiate into effector phenotypes such as Th1 (secreting IFN-gamma) or Th2 (secreting IL-4) that mediate effective immunity ...against different classes of pathogen. However, primed CD4 T cells can also remain uncommitted, secreting IL-2 and chemokines, but not IFN-gamma or IL-4. We now show that human CD4 T cells primed by protein vaccines mostly secreted IL-2, but not IFN-gamma, whereas in the same individuals most CD4 T cells initially primed by infection with live pathogens secreted IFN-gamma. We further demonstrate that many tetanus-specific IL-2+IFN-gamma- cells are uncommitted and that a single IL-2+IFN-gamma- cell can differentiate into Th1 or Th2 phenotypes following in vitro stimulation under appropriate polarizing conditions. In contrast, influenza-specific IL-2+IFN-gamma- CD4 cells maintained a Th1-like phenotype even under Th2-polarizing conditions. Similarly, adoptively transferred OTII transgenic mouse T cells secreted mainly IL-2 after priming with OVA in alum, but were biased toward IFN-gamma secretion when primed with the same OVA peptide presented as a pathogen Ag during live infection. Thus, protein subunit vaccines may prime a unique subset of differentiated, but uncommitted CD4 T cells that lack some of the functional properties of committed effectors induced by infection. This has implications for the design of more effective vaccines against pathogens requiring strong CD4 effector T cell responses.
Alloantigen-stimulated CD8
+ mouse spleen cells, either spontaneously or in the presence of IL-12 or IFNγ plus anti-IL-4, differentiate into CD8
+ T cells secreting a Th1-like cytokine pattern (IL-2 ...and IFNγ). IL-4 induced differentiation into CD8
+ T cells secreting Th2 cytokines (IL-4, IL-5, IL-6, and IL-10), whereas anti-IFNγ suppressed the development of CD8
+ cells secreting IFNγ. Clones of IL-4- or IFNγ-producing CD8
+ T cells were relatively stable, as IL-4 or IFNγ did not cause interconversion of committed CD8
+ T cells. Both CD8
+ subsets were cytotoxic, failed to provide cognate help for B cell antibody production, and remained CD4
−, CD8α
+CD8β
+. We propose the names TC1 and TC2 for cytotoxic CD8
+ T cells secreting Th1-like and Th2-like cytokines, respectively.
Within overall Th1-like human memory T cell responses, individual T cells may express only some of the characteristic Th1 cytokines when reactivated. In the Th1-oriented memory response to influenza, ...we have tested the contributions of two potential mechanisms for this diversity: variable expression of cytokines by a uniform population during activation, or different stable subsets that consistently expressed subsets of the Th1 cytokine pattern. To test for short-term variability, in vitro-stimulated influenza-specific human memory CD4+ T cells were sorted according to IL-2 and IFNgamma expression, cultured briefly in vitro, and cytokine patterns measured after restimulation. Cells that were initially IFNgamma+ and either IL-2+ or IL-2- converged rapidly, containing similar proportions of IL-2-IFNgamma+ and IL-2+IFNgamma+ cells after culture and restimulation. Both phenotypes expressed Tbet, and similar patterns of mRNA. Thus variability of IL-2 expression in IFNgamma+ cells appeared to be regulated more by short-term variability than by stable differentiated subsets. In contrast, heterogeneous expression of IFNgamma in IL-2+ influenza-specific T cells appeared to be due partly to stable T cell subsets. After sorting, culture and restimulation, influenza-specific IL-2+IFNgamma- and IL-2+IFNgamma+ cells maintained significantly biased ratios of IFNgamma+ and IFNgamma- cells. IL-2+IFNgamma- cells included both Tbet.sup.lo and Tbet.sup.hi cells, and showed more mRNA expression differences with either of the IFNgamma+ populations. To test whether IL-2+IFNgamma-Tbet.sup.lo cells were Thpp cells (primed but uncommitted memory cells, predominant in responses to protein vaccines), influenza-specific IL-2+IFNgamma- and IL-2+IFNgamma+ T cells were sorted and cultured in Th1- or Th2-generating conditions. Both cell types yielded IFNgamma-secreting cells in Th1 conditions, but only IL-2+IFNgamma- cells were able to differentiate into IL-4-producing cells. Thus expression of IL-2 in the anti-influenza response may be regulated mainly by short term variability, whereas different T cell subsets, Th1 and Thpp, may contribute to variability in IFNgamma expression.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Abstract The Elispot effectively measures the frequencies of cells secreting particular molecules, especially low-frequency cells such as antigen-specific T cells. The Fluorospot assay adapted this ...analysis to two products per cell, and this has now been extended to three-color measurement of both mouse and human cytokine-secreting cells. Due to the increased data complexity, and particularly the need to define single-, double- and triple-producing cells, it is critical to objectively quantify spot number, size, intensity, and coincidence with other spots. An automated counting program, Exploraspot, was therefore developed to detect and quantify Fluorospots in automated fluorescence microscope images. Morphological parameters, including size, intensity, location, circularity and others are calculated for each spot, exported in FCS format, and further analyzed by gating and graphical display in popular flow cytometry analysis programs. The utility of Exploraspot is demonstrated by identification of single-, double- and triple-secreting T cells; tolerance of variable background fluorescence; and estimation of the numbers of genuine versus random multiple events.
CD8(+) T cells in HIV-infected patients are believed to contribute to the containment of the virus and the delay of disease progression. However, the frequencies of HIV-specific CD8(+) T cells, as ...measured by IFN-gamma secretion and tetramer binding, often do not correlate with a delay in disease progression during chronic infection. Using the Lysispot and ELISPOT assays, we measured the frequencies of cytotoxic and IFN-gamma-secreting T cells responding to overlapping peptides from Gag, Nef, Env, and Pol consensus HIV-1 clade B sequences. PBMC from the majority of HIV-infected subjects have significant frequencies of HIV-specific cells that killed targets within 5 h directly ex vivo. The relative frequencies of IFN-gamma-secreting and cytotoxic cells varied markedly between different HIV peptide pools within the same patient, and some T cells lysed targets without secreting IFN-gamma. These results indicate that measurement of IFN-gamma production alone may be insufficient to evaluate the breadth of the HIV-specific T cell response. Also, neither the CTL to IFN-gamma ratios nor the ex vivo CTL frequencies specific for different HIV proteins were consistently lower than responses specific for two other chronic viral infections, human CMV and EBV, within the same subjects. Thus ex vivo cytotoxic T cell frequencies do not provide evidence for a model of "preterminal differentiation" of HIV-specific CD8(+) T cells during chronic HIV infection. Analysis of the frequency of directly cytotoxic HIV-specific T cells may be of considerable value in the assessment of disease progression and the potential efficacy of HIV vaccines.
Clinical and experimental evidence has indicated that the maternal immune response is biased toward antibody production and away from cell-mediated immunity during pregnancy, especially in the ...vicinity of the fetoplacental unit. Because antibody responses are often associated with the Th2 cytokine pattern, this suggests that Th2-type cytokines might predominate locally in the regulation of the maternal immune response. In order to test this hypothesis, we examined the local and distal release of cytokines during murine pregnancy using ELISA assays. We report here that the Th2-specific cytokines IL-4, IL-5, and IL-10 were readily detectable in cell supernatants derived from fetal-placental units in all three trimesters of gestation. IL-3 was also present. These cytokines were detected in lysates of freshly isolated, day 12 decidual and placental cells, and in supernatants as early as 15 min after the beginning of culture. The presence of functional IL-10 was confirmed by specific bioassay. IL-10 mRNA was localized to the decidua at day 6 of gestation by in situ hybridization. IFN-gamma was also found in the supernatants from the first trimester of pregnancy, but was barely detectable in the second, and undetectable in the third trimester. Cytokine expression was consistently detected in samples from individual mice. None of these cytokines was produced by unstimulated spleen or mesenteric lymph nodes from pregnant mice. IL-4, IL-10, and IFN-gamma were produced by Con A-stimulated spleen cells from virgin mice, but in ratios opposite to those found in the placenta. These observations indicate that Th2-specific cytokines are normally produced at the maternal-fetal interface. The continuous presence of IL-4, IL-5, and IL-10, with early and transient expression of IFN-gamma, can provide a molecular basis for the antibody/Th2-like bias of the maternal immune response during pregnancy.
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