Competitive SWIFT cluster templates enhance detection of aging changes.

Clustering-based algorithms for automated analysis of flow cytometry datasets have achieved more efficient and objective analysis than manual processing. Clustering organizes flow cytometry data into subpopulations with substantially homogenous characteristics but does not directly address the important problem of identifying the salient differences in subpopulations between subjects and groups. Here, we address this problem by augmenting SWIFT--a mixture model based clustering algorithm reported previously. First, we show that SWIFT clustering using a "template" mixture model, in which all subpopulations are represented, identifies small differences in cell numbers per subpopulation between samples. Second, we demonstrate that resolution of inter-sample differences is increased by "competition" wherein a joint model is formed by combining the mixture model templates obtained from different groups. In the joint model, clusters from individual groups compete for the assignment of cells, sharpening differences between samples, particularly differences representing subpopulation shifts that are masked under clustering with a single template model. The benefit of competition was demonstrated first with a semisynthetic dataset obtained by deliberately shifting a known subpopulation within an actual flow cytometry sample. Single templates correctly identified changes in the number of cells in the subpopulation, but only the competition method detected small changes in median fluorescence. In further validation studies, competition identified a larger number of significantly altered subpopulations between young and elderly subjects. This enrichment was specific, because competition between templates from consensus male and female samples did not improve the detection of age-related differences. Several changes between the young and elderly identified by SWIFT template competition were consistent with known alterations in the elderly, and additional altered subpopulations were also identified. Alternative algorithms detected far fewer significantly altered clusters. Thus SWIFT template competition is a powerful approach to sharpen comparisons between selected groups in flow cytometry datasets.

Long-term expansion of effector/memory Vdelta2-gammadelta T cells is a specific blood signature of CMV infection.

The ability of human gammadelta T cells to develop immunologic memory is still a matter of debate. We previously demonstrated the involvement of Vdelta2- gammadelta T lymphocytes in the response of immunosuppressed organ recipients to cytomegalovirus (CMV). Here, we demonstrate their ability to mount an adaptive immune response to CMV in immunocompetent subjects. Vdelta2- gammadelta T-cell peripheral blood numbers, repertoire restriction, and cytotoxicity against CMV-infected fibroblasts were markedly increased in CMV-seropositive, compared with CMV-seronegative, healthy persons. Whereas Vdelta2- gammadelta T cells were found as naive cells in CMV- patients, they virtually all exhibited the cytotoxic effector/memory phenotype in CMV+ patients, which is also observed in transplanted patients challenged with CMV. This long-term complete remodeling of the Vdelta2- gammadelta T-cell population by CMV predicts their ability to exhibit an adaptive anti-CMV immune response. Consistent with this, we observed that the secondary response to CMV was associated with a faster gammadelta T-cell expansion and a better resolution of infection than the primary response. In conclusion, the increased level of effector-memory Vdelta2- gammadelta T cells in the peripheral blood is a specific signature of an adaptive immune response to CMV infection of both immunocompetent and immunosuppressed patients.

Unbiased analysis of peripheral blood mononuclear cells reveals CD4 T cell response to RSV matrix protein.

Respiratory syncytial virus (RSV) is the most important cause of respiratory tract illness especially in young infants that develop severe disease requiring hospitalization, and accounting for 74,000-126,000 admissions in the United States (Rezaee et al., 2017; Resch, 2017). Observations of neonatal and infant T cells suggest that they may express different immune markers compared to T-cells from older children. Flow cytometry analysis of cellular responses using "conventional" anti-viral markers (IL2, IFN-γ, TNF, IL10 and IL4) upon RSV-peptide stimulation detected an overall low RSV response in peripheral blood. Therefore we sought an unbiased approach to identify RSV-specific immune markers using RNA-sequencing upon stimulation of infant PBMCs with overlapping peptides representing RSV antigens. To understand the cellular response using transcriptional signatures, transcription factors and cell-type specific signatures were used to investigate breadth of response across peptides. Unexpected from the ICS data, M peptide induced a response equivalent to the F-peptide and was characterized by activation of GATA2, 3, STAT3 and IRF1. This along with upregulation of several unconventional T cell signatures was only observed upon M-peptide stimulation. Moreover, signatures of natural RSV infections were identified from the data available in the public domain to investigate similarities between transcriptional signatures from PBMCs and upon peptide stimulation. This analysis also suggested activation of T cell response upon M-peptide stimulation. Hence, based on transcriptional response, markers were chosen to validate the role of M-peptide in activation of T cells. Indeed, CD4+CXCL9+ cells were identified upon M-peptide stimulation by flow cytometry. Future work using additional markers identified in this study could reveal additional unconventional T cells responding to RSV infections in infants. In conclusion, T cell responses to RSV in infants may not follow the canonical Th1/Th2 patterns of effector responses but include additional functions that may be unique to the neonatal period and correlate with clinical outcomes.

Modulation of lymphocyte proliferation induced by gastric MALT lymphoma-associated Helicobacter pylori strains.

BACKGROUND: Helicobacter pylori infection leads to different chronic diseases, suggesting that this bacterium can evade the host immune defense system. The ability to control lymphocyte proliferation may be a mechanism leading to the development of gastric pathologies. Our aim was to characterize the effects of mucosa-associated lymphoid tissue (MALT) associated H. pylori strains on lymphocyte proliferation. MATERIALS AND METHODS: We measured the in vitro proliferation of human lymphocytes originally from blood or tonsil samples in the presence or absence of viable bacteria or lysates. RESULTS: We showed that MALT lymphoma-associated strains are not likely to be directly responsible for anarchical B-cell proliferation in vitro. On the other hand, proliferation of prestimulated T lymphocytes was abolished in vitro by the presence of all H. pylori strains, whether associated with MALT lymphoma or not. CONCLUSION: Inhibition of T-cell proliferation may be of major importance in the gastric colonization and in the persistence of the infection. Furthermore, this inhibition may favor anarchical B-cell proliferation in vivo and predispose the host to gastric MALT lymphoma, whereas MALT-associated H. pylori strains do not appear to possess a specific capability to directly stimulate B-lymphocyte proliferation.

CD4+ T-cell responses among adults and young children in response to Streptococcus pneumoniae and Haemophilus influenzae vaccine candidate protein antigens.

We characterized cytokine profiles of CD4(+) T-helper (h) cells in adults and young children to ascertain if responses occur to next-generation candidate vaccine antigens PspA, PcpA, PhtD, PhtE, Ply, LytB of Streptococcus pneumonia (Spn) and protein D and OMP26 of non-typeable Haemophilus influenzae (NTHi). Adults had vaccine antigen-specific Th1 and Th2 cells responsive to all antigens evaluated whereas young children had significant numbers of vaccine antigen-specific CD4(+) T cells producing IL-2, (p=0.004). Vaccine antigen-specific CD4(+) T-cell populations in adults were largely of effector (TEM) and/or central memory (TCM) phenotypes as defined by CD45RA(-)CCR7(+) or CD45RA(-)CCR7(-) respectively; however among young children antigen-specific IL-2 producing CD4(+) T cells demonstrated CD45RA(+) expression (non-memory cells). We conclude that adults have circulating memory CD4(+) T cells (CD45RA(-)) that can be stimulated by all the tested Spn and NTHi protein vaccine candidate antigens, whereas young children have a more limited response.

Increase in IFNγ(-)IL-2(+) cells in recent human CD4 T cell responses to 2009 pandemic H1N1 influenza.

Human CD4 T cell recall responses to influenza virus are strongly biased towards Type 1 cytokines, producing IFNγ, IL-2 and TNFα. We have now examined the effector phenotypes of CD4 T cells in more detail, particularly focusing on differences between recent versus long-term, multiply-boosted responses. Peptides spanning the proteome of temporally distinct influenza viruses were distributed into pools enriched for cross-reactivity to different influenza strains, and used to stimulate antigen-specific CD4 T cells representing recent or long-term memory. In the general population, peptides unique to the long-circulating influenza A/New Caledonia/20/99 (H1N1) induced Th1-like responses biased toward the expression of IFNγ(+)TNFα(+) CD4 T cells. In contrast, peptide pools enriched for non-cross-reactive peptides of the pandemic influenza A/California/04/09 (H1N1) induced more IFNγ(-)IL-2(+)TNFα(+) T cells, similar to the IFNγ(-)IL-2(+) non-polarized, primed precursor T cells (Thpp) that are a predominant response to protein vaccination. These results were confirmed in a second study that compared samples taken before the 2009 pandemic to samples taken one month after PCR-confirmed A/California/04/09 infection. There were striking increases in influenza-specific TNFα(+), IFNγ(+), and IL-2(+) cells in the post-infection samples. Importantly, peptides enriched for non-cross-reactive A/California/04/09 specificities induced a higher proportion of Thpp-like IFNγ(-)IL-2(+)TNFα(+) CD4 T cells than peptide pools cross-reactive with previous influenza strains, which induced more Th1 (IFNγ(+)TNFα(+)) responses. These IFNγ(-)IL-2(+)TNFα(+) CD4 T cells may be an important target population for vaccination regimens, as these cells are induced upon infection, may have high proliferative potential, and may play a role in providing future effector cells during subsequent infections.

Expression of MHC class I receptors confers functional intraclonal heterogeneity to a reactive expansion of gammadelta T cells.

NK cell receptors for MHC class I molecules (MHC-NKR) can be expressed by T cell subsets. The restricted repertoire and phenotypic characteristics of MHC-NKR(+) T cells indicate that expression of MHC-NKR is acquired upon antigenic challenge and might promote expansion of T cells. Previous studies performed on in vitro generated alphabeta T cell clones concluded that MHC-NKR expression was not a clonal attribute. Here, we examined a massive monoclonal expansion of a non-leukemic gammadelta T cell population found in the peripheral blood of a lung-transplanted patient who suffered from a cytomegalovirus infection. Despite their monoclonality, these T cells displayed a heterogeneous and stable in vivo Ig- and lectin-like MHC-NKR phenotype. Twenty percent of the cells displayed a CD94(+)NKG2A(+) phenotype, and 10% were labeled with an anti-CD158b1/b2/j monoclonal antibody. A CD158b/j(+) gammadelta T cell clone derived in vitro from patient's peripheral blood lymphocytes was shown to express the activating form CD158j (KIR2DS2), which once cross-linked stimulated the clone cytolytic function and costimulated the TCR-induced production of cytokines, independently of the killer-activating receptor-associated protein (KARAP). In conclusion, heterogeneity of MHC-NKR expression confers a functional intraclonal diversity that may participate to induction of specific gammadelta T cell effector functions or proliferation upon pathogen challenge.