O+ uninfected RBCs (uRBC) were used as controls. using FSC and SSC to select lymphocytes, then by excluding DUMP (CD14, CD19, Aqua live/dead) positive cells, and finally by selecting for CD3 positive and V2 positive events. uRBC or isotype controls were used to define IFN and CD107a positive events, as shown.(TIF) ppat.1008997.s003.tif (1.3M) GUID:?5B60F244-200E-425A-B29E-D246855904C1 S3 Fig: CD16+ V2 T cells are more likely to express cytotoxic markers regardless of age and exposure history. (A) The percentage of CD16+ and CD16-V2 T cells expressing relevant cytotoxic markers, divided by age (children vs adults) and exposure history (high exposure = Nagogera, low exposure = Walakuba) (Walakuba children, n = SB1317 (TG02) 20; Nagongera children, n = 20; Walakuba adults, n = 13; Nagongera adults, n = 12). Scatter plots with median and IQR are shown. P values were determined by Wilcoxon matched pairs signed rank test.(TIF) ppat.1008997.s004.tif IKK-gamma antibody (3.4M) GUID:?3228B11D-61FC-4A77-AAFA-23809FD126E0 S4 Fig: Hyper-immune IgG opsonizes 3D7 in an innate-like manner, without prior antigen exposure or processing. V2 T cells have been shown to inhibit parasite replication and are associated with protection from parasitemia infection. A better understanding of how these cells interact with malaria parasites to control infection is necessary. We have previously shown that after multiple infections, V2 T cells decrease in frequency, become less responsive to TCR stimulation, and upregulate the Fc receptor CD16. Here we investigate whether V2 T cells from chronically malaria-exposed individuals can be activated directly through CD16 to release proinflammatory cytokines and degranulate. We show that in these individuals, TCR is downregulated on CD16+ V2 T cells, and that these cells are more likely to express a variety of cytotoxic effector molecules. Importantly, we show that these CD16+ V2 T cells can be activated directly through CD16, independent of TCR, by antibody bound to parasite antigen. These results are notable because they indicate many V2 T cells from chronically-exposed individuals may not be exhausted but instead favor an alternative activation pathway, one that cooperates with a mature anti-malarial antibody response. Introduction T cells are believed to play an important role in the immune response to malaria. These unconventional lymphocytes comprise up to 5% of peripheral blood T cells and exhibit features of both adaptive and innate immune cells. T cells expressing the V2 and V9 TCR chains are intrinsically reactive to malaria due to their activation by low-molecular weight phosphoantigens produced by the apicoplast. V9V2 T cells recognize malaria-derived phosphoantigens such as HMBPP with exquisite sensitivity via a unique mode SB1317 (TG02) of presentation [1C5]. Phosphoantigens bind to the conserved transmembrane presenting molecules Butyrophylin 3A1 (BTN3A1) [6] and Butyrophilin 2A1 [7,8], inducing a conformational shift that is sensed via cognate interaction with the V9V2 TCR. Because BTN3a1 is ubiquitously expressed, this interaction is MHC-unrestricted and does not require professional antigen presenting cells. Thus, V2 T cells act as innate-like effectors that can be rapidly activated during primary infection before an adaptive response has developed. Indeed, massive expansions of V2 T cells have been reported during acute malaria in previously na?ve hosts [9C12]. V2 T cells inhibit replication of blood-stage parasites by the release of cytotoxic granules containing granulysin [4,13]. The frequency and malaria-responsiveness of V2 T cells has been shown to correlate with protection from parasitemia in naturally exposed Ugandan children and in malaria-naive volunteers immunized with attenuated sporozoites [14C16]. In contrast to acute malaria, chronic exposure to malaria is associated with a decline in both the frequency of V2 T cells and their ability to produce pro-inflammatory cytokines in response to antigen. This declining frequency of malaria-reactive V2 T cells has been associated with a lower likelihood of symptoms upon subsequent infection [17]. Chronic malaria exposure results in numerous transcriptional changes in V2 T cells [17]. Among these is expression of the Fc receptor CD16/FcRIIIa, which is markedly upregulated in the setting of frequent malaria exposure, but is expressed at low levels in children with little or no prior malaria [18]. We have previously shown that CD16/FcRIIIa expression identifies a subset of V2 T cells that are largely unresponsive to stimulation with antigen [18]. A notable feature of T cells is their functional plasticity. Prior studies indicate that CD16 discriminates functionally distinct subsets of V2 cells, and that direct ligation of CD16 may provide an alternate pathway of V2 activation [19]. Activation of T cells through CD16 by opsonized antigen has been shown to mediate antibody-dependent cell-mediated cytotoxicity [20], phagocytosis [21], cytokine release [22], and licensing for professional antigen presentation [23,24]. SB1317 (TG02) The functional significance of the CD16 signaling pathway in V2 T cells has not been investigated in the setting.
