DNA and modified vaccinia Ankara prime-boost vaccination generates strong CD8+ T cell responses against minor histocompatibility antigen HA-1.

Allogeneic immune responses underlie the graft-versus-leukaemia effect of stem cell transplantation, but disease relapse occurs in many patients. Minor histocompatibility antigen (mHAg) peptides mediate alloreactive T cell responses and induce graft-versus-leukaemia responses when expressed on patient haematopoietic tissue. We vaccinated nine HA-1-negative donors against HA-1 with a 'prime-boost' protocol of either two or three DNA 'priming' vaccinations prior to 'boost' with modified vaccinia Ankara (MVA). HA-1-specific CD8+ T cell responses were observed in seven donors with magnitude up to 1·5% of total CD8+ T cell repertoire. HA-1-specific responses peaked two weeks post-MVA challenge and were measurable in most donors after 12 months. HA-1-specific T cells demonstrated strong cytotoxic activity and lysed target cells with endogenous HA-1 protein expression. The pattern of T cell receptor (TCR) usage by HA-1-specific T cells revealed strong conservation of T cell receptor beta variable 7-9 (TRBV7-9) usage between donors. These findings describe one of the strongest primary peptide-specific CD8+ T cell responses yet recorded to a DNA-MVA prime-boost regimen and this may reflect the strong immunogenicity of mHAg peptides. Prime-boost vaccination in donors or patients may prove of substantial benefit in boosting graft-versus-leukaemia responses.

Progesterone promotes maternal-fetal tolerance by reducing human maternal T-cell polyfunctionality and inducing a specific cytokine profile.

Progesterone is a steroid hormone essential for the maintenance of human pregnancy, and its actions are thought to include promoting maternal immune tolerance of the semiallogenic fetus. We report that exposure of maternal T cells to progesterone at physiological doses induced a unique skewing of the cytokine production profile of CD4(+) and CD8(+) T cells, with reductions not only in potentially deleterious IFN-γ and TNF-α production but also in IL-10 and IL-5. Conversely, production of IL-4 was increased. Maternal T cells also became less polyfunctional, focussing cytokine production toward profiles including IL-4. This was accompanied by reduced T-cell proliferation. Using fetal and viral antigen-specific CD8(+) T-cell clones, we confirmed that this as a direct, nonantigen-specific effect. Yet human T cells lacked conventional nuclear progesterone receptors, implicating a membrane progesterone receptor. CD4(+) and CD8(+) T cells responded to progesterone in a dose-dependent manner, with subtle effects at concentrations comparable to those in maternal blood, but profound effects at concentrations similar to those at the maternal-fetal interface. This characterization of how progesterone modulates T-cell function is important in understanding the normal biology of pregnancy and informing the rational use of progesterone therapy in pregnancies at risk of fetal loss.

Chemokine-mediated tissue recruitment of CXCR3+ CD4+ T cells plays a major role in the pathogenesis of chronic GVHD.

Chemokines regulate the migration of hemopoietic cells and play an important role in the pathogenesis of many immune-mediated diseases. Intradermal recruitment of CD8(+) T cells by CXCL10 is a central feature of the pathogenesis of cutaneous acute GVHD (aGVHD), but very little is known about the pathogenesis of chronic GVHD (cGVHD). Serum concentrations of the 3 CXCR3-binding chemokines, CXCL9, CXCL10, and CXCL11, were found to be markedly increased in patients with active cGVHD of the skin (n = 8). An 80% decrease in CD4(+) cells expressing CXCR3 was seen in the blood of these patients (n = 5), whereas CD4(+) cells were increased in tissue biopsies and were clustered around the central arterioles of the dermis. The well-documented increase in expression of CXCL10 in aGVHD therefore diversifies in cGVHD to include additional members of the CXCR3-binding family and leads to preferential recruitment of CD4(+) T cells. These observations reveal a central role for chemokine-mediated recruitment of CXCR3(+) T cells in cGVHD.

Most B cells in non-lymphoid tissues are naïve.

The current view of lymphocyte migration states that naïve lymphocytes re-circulate between the blood and the lymph via the lymph nodes, but are not able to access non-lymphoid tissues. We examined B lymphocytes in peripheral tissues and found that the majority were phenotypically similar to naïve B cells in lymphoid tissues and were located within the parenchyma, not associated with blood vessels. The mutation rate within the Vh region of these cells was substantially less than the rate attributed to somatic hypermutation and was identical to that observed in naïve B cells isolated from the lymph nodes, showing the presence of naïve B cells in the non-lymphoid organs. Further, using FTY720-treated mice, we showed that naïve B cells migrate through the peripheral tissues and, using pertussis toxin, that the entry of B cells was not controlled by chemokine-mediated signalling events. Overall, these results show that naïve B lymphocytes constitute the majority of the total B-cell population in non-lymphoid tissues and suggest that these cells may re-circulate through the periphery as part of their normal migration pathway. This has implications for the current view of the role of naïve B cells in priming and tolerance.

Direct experimental evidence that early-life farm environment influences regulation of immune responses.

BACKGROUND: In mammals, early-life environmental variations appear to affect microbial colonization and therefore competent immune development, and exposure to farm environments in infants has been inversely correlated with allergy development. Modelling these effects using manipulation of neonatal rodents is difficult due to their dependency on the mother, but the relatively independent piglet is increasingly identified as a valuable translational model for humans. This study was designed to correlate immune regulation in piglets with early-life environment. METHODS: Piglets were nursed by their mother on a commercial farm, while isolator-reared siblings were formula fed. Fluorescence immunohistology was used to quantify T-reg and effector T-cell populations in the intestinal lamina propria and the systemic response to food proteins was quantified by capture ELISA. RESULTS: There was more CD4(+) and CD4(+) CD25(+) effector T-cell staining in the intestinal mucosa of the isolator-reared piglets compared with their farm-reared counterparts. In contrast, these isolator-reared piglets had a significantly reduced CD4(+) CD25(+) Foxp3(+) regulatory T-cell population compared to farm-reared littermates, resulting in a significantly higher T-reg-to-effector ratio in the farm animals. Consistent with these findings, isolator-reared piglets had an increased serum IgG anti-soya response to novel dietary soya protein relative to farm-reared piglets. CONCLUSION: Here, we provide the first direct evidence, derived from intervention, that components of the early-life environment present on farms profoundly affects both local development of regulatory components of the mucosal immune system and immune responses to food proteins at weaning. We propose that neonatal piglets provide a tractable model which allows maternal and treatment effects to be statistically separated.

CD52/GPI- T-Cells Are Enriched for Alloreactive Specificity and Predict Acute Graft-Versus-Host-Disease After Stem Cell Transplantation.

Alemtuzumab is a CD52-specific lympho-depleting antibody. CD52- T cells emerge under alemtuzumab selection pressure. We sought to investigate the phenotype and function of the CD52- T cell fraction and related their presence to clinical outcome. We obtained longitudinal peripheral blood samples from 67 consecutive patients undergoing allo-HSCT between 2013-2016. Forty-seven patients (70%) had a myeloid disease (acute myelogenous leukemia or myelodysplastic syndrome) whereas 20 patients had lymphoid disease. All patients received in vivo alemtuzumab (10 mg/d from day -5 for 5 days) as part of their conditioning protocol. Sixty-three (94%) received reduced-intensity conditioning chemotherapy, whereas 4 (6%) received a myeloablative regimen. All patients received post-transplantation cyclosporine A for graft-versus-host disease (GVHD) prophylaxis. Six (9%) also received methotrexate, whereas 2 (3%) patients also received mycophenolate mofetil. Overall survival at 2 years was 68%, and relapse-free survival was 48%. Twenty-none percent of patients experienced acute GVHD (grade 2 or above), and 15% developed chronic GVHD. CD52- T cells were detectable in 66 of 67 consecutive patients. CD52- T cells demonstrated low binding of fluorescent aerolysin, indicating downregulation of the glycophosphatidylinositol anchor, although we did not detect any mutations in the PIG-A gene as is typically seen in patients with paroxysmal nocturnal hemoglobinuria. CD52- T cells were almost exclusively CD4+ and exhibited a dominant memory phenotype with only small numbers of CD25+ CD127low Foxp3+ regulatory T cells. CD52- T cells exhibited alloreactive specificity in vitro and have a distinct TCR repertoire to CD52+ T cells. Early after allo-hematopoietic stem cell transplantation, the presence of a significant population of CD52- T cells (comprising >51% of the T cell fraction) was found to be an independent risk factor for acute GvHD. This was confirmed in a validation cohort of 28 patients obtained between 2017-2018. These data suggest that the CD52- T cell fraction may represent a residual "footprint" of an early CD4+ T cell alloreactive response and may have been rescued from alemtuzumab-mediated lysis by antigen engagement in vivo. These data help to delineate the nature of T cell escape from alemtuzumab surveillance and contribute to increasing interest in the importance of CD4+ T cells in alloreactive immune responses, which could help inform immunotherapy protocols.

The mucosal immune response to laryngopharyngeal reflux.

RATIONALE: Laryngopharyngeal reflux (LPR) affects up to 20% of Western populations. Although individual morbidity is usually moderate, treatment costs are high and there are associations with other diseases, including laryngeal cancer. To date, there have been no studies of the mucosal immune response to this common inflammatory disease. OBJECTIVES: To determine the mucosal immune response to LPR. METHODS: We performed a prospective immunologic study of laryngeal biopsies from patients with LPR and control subjects (n = 12 and 11, respectively), and of primary laryngeal epithelial cells in vitro. MEASUREMENTS AND MAIN RESULTS: Quantitative multiple-color immunofluorescence, using antibodies for lymphocytes (CD4, CD8, CD3, CD79, CD161), granulocytes (CD68, EMBP), monocytic cells (CD68, major histocompatibility complex [MHC] class II), and classical and nonclassical MHC (I, II, beta(2)-microglobulin, CD1d). Univariate and multivariate analysis and colocalization measurements were applied. There was an increase in percentage area of mucosal CD8(+) cells in the epithelium (P < 0.005), whereas other leukocyte and granulocyte antigens were unchanged. Although epithelial MHC class I and II expression was unchanged by reflux, expression of the nonclassical MHC molecule CD1d increased (P < 0.05, luminal layers). In vitro, laryngeal epithelial cells constitutively expressed CD1d. CD1d and MHC I expression were inversely related in all subjects, in a pattern which appears to be unique to the upper airway. Colocalization of natural killer T (NKT) cells with CD1d increased in patients (P < 0.01). CONCLUSIONS: These data indicate a role for the CD1d-NKT cell axis in response to LPR in humans. This represents a useful target for novel diagnostics and treatments in this common condition.

Mixed chimerism established by hematopoietic stem cell transplantation is maintained by host and donor T regulatory cells.

Transplantation is an effective treatment of many clinical disorders, but the mechanisms that regulate immunological tolerance are uncertain and remain central to improving patient outcome. Hemopoietic stem cell transplantation (SCT) often establishes "mixed chimerism" in which immune cells from both the donor and patient coexist in vivo in a setting of immunological tolerance. We studied immune function in 69 patients within 2 months following SCT; 37 were fully donor and 32 displayed mixed chimerism. The proportion of T regulatory (Treg) cells was increased during mixed chimerism and comprised equal numbers of donor and host-derived regulatory cells. This was associated with a tolerogenic PD-L1+ profile on dendritic cells. Importantly, effector T cells from patients with mixed chimerism exhibited reduced cytotoxicity against host target cells in vitro, but this was restored following depletion of CD4+ Treg cells. These data show that Treg cells play a major role in sustaining immunological tolerance during mixed chimerism. These insights should help to guide novel interventions to improve clinical transplantation.

Very early lineage-specific chimerism after reduced intensity stem cell transplantation is highly predictive of clinical outcome for patients with myeloid disease.

BACKGROUND: The importance of chimerism status in the very early period after hematopoietic stem cell transplantation is unclear. We determined PBMC and T-cell donor chimerism 50 days after transplantation and related this to disease relapse and overall survival. METHODS: 144 sequential patients underwent transplantation of which 90 had AML/MDS and 54 had lymphoma. 'Full donor chimerism' was defined as ≥99% donor cells and three patient groups were defined: 40% with full donor chimerism (FC) in both PBMC and T-cells; 25% with mixed chimerism (MC) within both compartments and 35% with 'split' chimerism (SC) characterised by full donor chimerism within PBMC and mixed chimerism within T-cells. RESULTS: In patients with myeloid disease a pattern of mixed chimerism (MC) was associated with a one year relapse rate of 45% and a five year overall survival of 40% compared to values of 8% and 75%, and 17% and 60%, for those with SC or FC respectively. The pattern of chimerism had no impact on clinical outcome for lymphoma. CONCLUSION: The pattern of lineage-specific chimerism at 50 days after transplantation is highly predictive of clinical outcome for patients with myeloid malignancy and may help to guide subsequent clinical management.

Immunologic response of the laryngeal mucosa to extraesophageal reflux.

OBJECTIVES: Extraesophageal reflux is common. The treatment costs are high, and there are associations with other diseases, including laryngeal cancer. Our studies of the mucosal immune response to this common inflammatory disease suggest an important role for the nonclassic antigen-presenting molecule CD1d in the response to inflammation. This study was performed to further explore the relationship between the CD1d-NKT cell-iGb3 axis and reflux. METHODS: We carried out a prospective study of laryngeal biopsies from 12 patients with laryngopharyngeal reflux and 11 controls. Quantitative multiple-color immunofluorescence using antibodies for lymphocytes (CD3, CD161) and classic and nonclassic major histocompatibility complex (I, II, beta2m, CD1d) was performed, and univariate and multivariate analysis and co-localization measurements were applied. RESULTS: Epithelial major histocompatibility complex class I and II expression was unchanged by reflux, but expression of CD1d increased (p < 0.05; luminal layers) and confidence intervals diminished in the reflux group. Co-localization of NKT cells with CD1d increased in patients (p < 0.01); iGb3 exhibited strong expression throughout all layers of the laryngeal epithelium. CONCLUSIONS: These data indicate a role for the CD1d-NKT cell-iGb3 axis in response to extraesophageal reflux in humans. This represents a useful target for novel diagnostics and treatments for this common condition.

Unique features and clinical importance of acute alloreactive immune responses.

Allogeneic stem cell transplantation (allo-SCT) can cure some patients with hematopoietic malignancy, but this relies on the development of a donor T cell alloreactive immune response. T cell activity in the first 2 weeks after allo-SCT is crucial in determining outcome, despite the clinical effects of the early alloreactive immune response often not appearing until later. However, the effect of the allogeneic environment on T cells is difficult to study at this time point due to the effects of profound lymphopenia. We approached this problem by comparing T cells at week 2 after allograft to T cells from autograft patients. Allograft T cells were present in small numbers but displayed intense proliferation with spontaneous cytokine production. Oligoclonal expansions at week 2 came to represent a substantial fraction of the established T cell pool and were recruited into tissues affected by graft-versus-host disease. Transcriptional analysis uncovered a range of potential targets for immune manipulation, including OX40L, TWEAK, and CD70. These findings reveal that recognition of alloantigen drives naive T cells toward a unique phenotype. Moreover, they demonstrate that early clonal T cell responses are recruited to sites of subsequent tissue damage and provide a range of targets for potential therapeutic immunomodulation.

Neonatal colonisation expands a specific intestinal antigen-presenting cell subset prior to CD4 T-cell expansion, without altering T-cell repertoire.

Interactions between the early-life colonising intestinal microbiota and the developing immune system are critical in determining the nature of immune responses in later life. Studies in neonatal animals in which this interaction can be examined are central to understanding the mechanisms by which the microbiota impacts on immune development and to developing therapies based on manipulation of the microbiome. The inbred piglet model represents a system that is comparable to human neonates and allows for control of the impact of maternal factors. Here we show that colonisation with a defined microbiota produces expansion of mucosal plasma cells and of T-lymphocytes without altering the repertoire of alpha beta T-cells in the intestine. Importantly, this is preceded by microbially-induced expansion of a signal regulatory protein α-positive (SIRPα(+)) antigen-presenting cell subset, whilst SIRPα(-)CD11R1(+) antigen-presenting cells (APCs) are unaffected by colonisation. The central role of intestinal APCs in the induction and maintenance of mucosal immunity implicates SIRPα(+) antigen-presenting cells as orchestrators of early-life mucosal immune development.

Immune responses in pigs vaccinated with adjuvanted and non-adjuvanted A(H1N1)pdm/09 influenza vaccines used in human immunization programmes.

Following the emergence and global spread of a novel H1N1 influenza virus in 2009, two A(H1N1)pdm/09 influenza vaccines produced from the A/California/07/09 H1N1 strain were selected and used for the national immunisation programme in the United Kingdom: an adjuvanted split virion vaccine and a non-adjuvanted whole virion vaccine. In this study, we assessed the immune responses generated in inbred large white pigs (Babraham line) following vaccination with these vaccines and after challenge with A(H1N1)pdm/09 virus three months post-vaccination. Both vaccines elicited strong antibody responses, which included high levels of influenza-specific IgG1 and haemagglutination inhibition titres to H1 virus. Immunisation with the adjuvanted split vaccine induced significantly higher interferon gamma production, increased frequency of interferon gamma-producing cells and proliferation of CD4(-)CD8(+) (cytotoxic) and CD4(+)CD8(+) (helper) T cells, after in vitro re-stimulation. Despite significant differences in the magnitude and breadth of immune responses in the two vaccinated and mock treated groups, similar quantities of viral RNA were detected from the nasal cavity in all pigs after live virus challenge. The present study provides support for the use of the pig as a valid experimental model for influenza infections in humans, including the assessment of protective efficacy of therapeutic interventions.

Dendritic cells interact with CD4 T cells in intestinal mucosa.

Absence of lymph nodes in nonmammalian species, expression of MHCII by APCs in the periphery, and the recent findings that T cells can change their polarization status after presentation in the lymph nodes imply a role for MHCII-mediated presentation outside the organized lymphoid tissue. This study shows that MHCII(+) ECs and DCs from the intestinal mucosa of the pig can present antigen to T cells in vitro. In vivo, APCs colocalize with T cells in pig and mouse intestinal mucosa. In the pig, endothelium is involved in these interactions in neonates but not in adults, indicating different roles for stromal and professional APCs in the neonate compared with the adult. The ratio of expression of DQ and DR MHCII locus products was lower on ECs than on other mucosal APCs, indicating that the two types of cells present different peptide sets. Adult nonendothelial APCs expressed a higher ratio of DQ/DR than in neonates. These results suggest that mucosal DCs can present antigen locally to primed T cells and that stromal APCs are recruited to these interactions in some cases. This raises the possibility that local presentation may influence T cell responses at the effector stage after initial presentation in the lymph node.