PBMC Responses Research Articles

High Dimensional Phenotypic and Functional Analysis of The Immune Response During Post-Transplant Lymphoproliferative Disorder Using Mass Cytometry.

Post-transplant lymphoproliferative disorder (PTLD) is a life-threatening complication of organ transplantation that is often linked to EBV infection and impaired host T cell responses. We utilized mass cytometry to achieve high dimensional analysis of the EBV-specific immune response during PTLD. PBMC were obtained prior to treatment from 10 PTLD patients (n=5 CNS-associated disease, n=5 peripheral diffuse large B cell lymphoma, average age=60, time to PTLD=13.7 years post-transplant). Using cytometry by time-of-flight (CyTOF) mass cytometry, we examined phenotype and cytokine responses of PBMC following stimulation with EBV lytic or latent peptides. Thirty-seven markers, both intracellular and surface, were analyzed simultaneously. In order to reduce the dimensionality of the dataset, cells were clustered using hierarchical agglomerative clustering on cell-surface markers. Changes in the percent of cells in each cluster upon stimulation with EBV peptides were determined and responses were grouped using principal component analysis (PCA). Four of 10 PTLD patients had increased numbers of an unusual population of NK-like CD16+CD56- cells and decreased numbers of T cells; in 6 patients T cells predominated. PBMC from PTLD patients showed a range of responses to both EBV lytic and latent peptides. IFNγ, MIP-1β and TNFα producing cells were detectable and the majority resembled T effector memory cells (CD4+CD45RA-CCR7- and CD8+CD45RA-CCR7-). The ratio of CD8+:CD4+ IFNγ+ cells responding to latent and lytic peptides was ˜1.6 in patients compared to the control ratio of 3.1. There was an increase in the percentage of CD56+ IFNγ+ cells in PTLD patients compared to control. Patients cluster separately from control using PCA for responses to both lytic and latent peptides. Thus, EBV+ PTLD is associated with alterations in lymphoid populations including increased proportions of CD16+CD56- NK-like cells and CD56+INFγ+ cells and a shift in the ratio of CD8+:CD4+ cytokine producing cells. Further characterization of immune cells in PTLD patients will be important for improving diagnosis and treatment of this disorder.

A pig tonsil cell culture model for evaluating oral, low-dose IFN-α treatments

Oral, low-dose IFN-α treatments proved effective in several models of viral infections and immunopathological conditions. Also, they do not give rise to the serious side effects observed after parenteral inoculation of high doses (105U/kg b.w. and higher). There is convincing evidence that such treatments work through an early, effective interaction with oral lymphoid tissues before the IFN-α molecules are rapidly destroyed by gut enzymes. Yet, the paucity of detailed information about these crucial interactions and the lack of recognized in vitro models hamper the development of proper administration protocols. On the basis of a previous study, we developed an in vitro model of interaction between different types of human and porcine IFNs-α at low/moderate concentrations and pig tonsil cells. The IFNs-α under study showed different properties with respect to three fundamental control actions: (1) IgA release in culture, (2) release of natural antimicrobial compounds, and (3) homeostatic regulation of the inflammatory response. This was checked in pig intestinal epithelial cells (IPEC-J2 cell line) treated with supernatants of control and IFN α-treated tonsil cell cultures, respectively, in terms of inflammatory cytokine and chemokine responses. Some IFNs-α caused a significant inhibition of IL-8 (protein release and gene expression) and beta-defensin 1 (gene expression) probably through second messengers released by IFN α-treated tonsil cells. Interestingly, a human lymphoblastoid IFN-α under study caused the decrease of polyclonal IgA release by pig tonsil cells and significantly stimulated the in vitro recall antibody response of swine PBMC to Foot-and-Mouth Disease virus. The modulation of IgA and antibacterial compounds was accompanied by an anti-inflammatory control action at the same, low to moderate IFN-α concentrations (1–100U/ml). This highlights the very foundation of the homeostatic control actions performed by Type I IFNs: to promote an effective host response to infectious and non-infectious stressors and to turn off noxious inflammatory responses associated with tissue damage and waste of metabolic energy. The described tonsil cell model in vitro can be conducive to a further development of oral cytokine treatments in humans and animals in the “one health” conceptual framework.

Innate immune correlates of HIV pathogenesis in response to opportunistic mycobacteria (INC8P.428)

Abstract HIV infection of humans and SIV infection of Rhesus macaques generally results in onset of opportunistic infections (OIs) and AIDS when left untreated. In contrast, SIV infection of African monkeys, such as sooty mangabeys, results in high levels of viral replication, but the immune system remains resilient to dysfunction, OIs and progression to AIDS. Here, we set out to identify innate immune correlates associated with acquisition of OIs in HIV+ humans (and the lack thereof in SIV+ mangabeys), through an ex vivo cytokine and transcriptomic assessment of PBMC and monocyte responses to the opportunistic pathogen, BCG. Our data reveal that HIV+ donors have significantly altered NK cell gene expression profiles and deficient monocyte IL-12 production (p=0.017) in response to BCG stimulation compared to HIV-negative donors. In contrast, SIV+ mangabeys maintain similar gene expression profiles and increase the percentage of monocytes producing proinflammatory cytokines TNFα (p=0.03) and IL-12 (p=0.06) following BCG stimulation compared to uninfected mangabeys. These data suggest that a lack of monocyte-derived IL-12 and deficient NK cell response to opportunistic pathogens may be associated with increased susceptibility to OIs. Further, they raise potential to augment innate immune function through NK cell-targeted IL-12 therapy during pathogenic lentiviral infections.

The IL-6 response to Chlamydia from primary reproductive epithelial cells is highly variable and may be involved in differential susceptibility to the immunopathological consequences of chlamydial infection

BackgroundChlamydia trachomatis infection results in reproductive damage in some women. The process and factors involved in this immunopathology are not well understood. This study aimed to investigate the role of primary human cellular responses to chlamydial stress response proteases and chlamydial infection to further identify the immune processes involved in serious disease sequelae.ResultsLaboratory cell cultures and primary human reproductive epithelial cultures produced IL-6 in response to chlamydial stress response proteases (CtHtrA and CtTsp), UV inactivated Chlamydia, and live Chlamydia. The magnitude of the IL-6 response varied considerably (up to 1000 pg ml-1) across different primary human reproductive cultures. Thus different levels of IL-6 production by reproductive epithelia may be a determinant in disease outcome. Interestingly, co-culture models with either THP-1 cells or autologous primary human PBMC generally resulted in increased levels of IL-6, except in the case of live Chlamydia where the level of IL-6 was decreased compared to the epithelial cell culture only, suggesting this pathway may be able to be modulated by live Chlamydia. PBMC responses to the stress response proteases (CtTsp and CtHtrA) did not significantly vary for the different participant cohorts. Therefore, these proteases may possess conserved innate PAMPs. MAP kinases appeared to be involved in this IL-6 induction from human cells. Finally, we also demonstrated that IL-6 was induced by these proteins and Chlamydia from mouse primary reproductive cell cultures (BALB/C mice) and mouse laboratory cell models.ConclusionsWe have demonstrated that IL-6 may be a key factor for the chlamydial disease outcome in humans, given that primary human reproductive epithelial cell culture showed considerable variation in IL-6 response to Chlamydia or chlamydial proteins, and that the presence of live Chlamydia (but not UV killed) during co-culture resulted in a reduced IL-6 response suggesting this response may be moderated by the presence of the organism.

Early inflammatory response to the saponin adjuvant Matrix-M in the pig

The early inflammatory response to Matrix-M was evaluated in pigs. Adverse reactions measured as body temperature, appetite, activity level and reaction at the site of injection were not observed after s.c. injection with three doses of the adjuvant (75, 100 or 150μg) into one week old piglets. Analyses of the immediate cytokine response of PBMC after in vitro exposure to Matrix-M (AbISCO-100®) revealed only a low expression of mRNA for tumour necrosis factor-α (p<0.05) after 6h incubation. Histological examination revealed an infiltration of leukocytes, haemorrhage and necrosis in muscle 24h after i.m. injection of 150μg Matrix-M in pigs aged eleven weeks. At this time, different grades of reactive lymphoid hyperplasia were recorded in the draining lymph node that was enlarged in three of these six pigs injected with Matrix-M. The global transcriptional response at the site of injection and in the draining lymph node was analyzed using Affymetrix GeneChip Porcine Genome Array. A significant enrichment of gene signatures for the cell types described as “myeloid cells” and “plasmacytoid dendritic cells” was observed at the site of injection in Matrix-M injected pigs compared with pigs injected with saline. A number of genes encoding cytokines/chemokines or their receptors were upregulated at the injection site as well as in the draining lymph node. In the draining lymph node, a majority of the upregulated genes were interferon-regulated genes (IRGs). The expression of IFN-β, but not IFN-α, was increased in the draining lymph nodes of a majority of the pigs exposed to Matrix-M. These IFN-β expressing pigs also expressed increased levels of osteopontin (OPN) or stimulator of interferon genes (STING), two factors known to facilitate the expression of type I IFNs in response to viral infection. Thus, Matrix-M does not appear to induce any harmful inflammatory response in piglets whilst contributing to the innate immunity by activating the type I IFN system, possibly through several alternative signalling pathways.

Comparison of Systemic and Mucosal Immunization with Helper-Dependent Adenoviruses for Vaccination against Mucosal Challenge with SHIV

Most HIV-1 infections are thought to occur at mucosal surfaces during sexual contact. It has been hypothesized that vaccines delivered at mucosal surfaces may mediate better protection against HIV-1 than vaccines that are delivered systemically. To test this, rhesus macaques were vaccinated by intramuscular (i.m.) or intravaginal (ivag.) routes with helper-dependent adenoviral (HD-Ad) vectors expressing HIV-1 envelope. Macaques were first immunized intranasally with species C Ad serotype 5 (Ad5) prior to serotype-switching with species C HD-Ad6, Ad1, Ad5, and Ad2 vectors expressing env followed by rectal challenge with CCR5-tropic SHIV-SF162P3. Vaccination by the systemic route generated stronger systemic CD8 T cell responses in PBMC, but weaker mucosal responses. Conversely, mucosal immunization generated stronger CD4 T cell central memory (Tcm) responses in the colon. Intramuscular immunization generated higher levels of env-binding antibodies, but neither produced neutralizing or cytotoxic antibodies. After mucosal SHIV challenge, both groups controlled SHIV better than control animals. However, more animals in the ivag. group had lower viral set points than in in the i.m. group. These data suggest mucosal vaccination may have improve protection against sexually-transmitted HIV. These data also demonstrate that helper-dependent Ad vaccines can mediate robust vaccine responses in the face of prior immunity to Ad5 and during four rounds of adenovirus vaccination.

The role of Th17+ T cells and neutrophils in mucosal immunity against Staphylococcus aureus mastitis. (P4256)

Abstract Staphylococcus aureus is a common cause of mastitis, a disease estimated to cost the dairy industry ~$2 billion/yr. Public concern over the use of antibiotics in agricultural animals necessitates the identification of new mechanisms for controlling this pathogen. Our goal is to define the role of immune activating DC in the development of protective S. aureus-specific T cell memory and use this knowledge for identification of antigens for vaccine development. Our findings indicate IL-17 generated by PBMC in response to S. aureus loaded monocyte-derived DC that is greater in memory as compared to naïve animals. Lymphocytes stimulated with both live and gamma irradiated antigens presented by monocyte-derived DC released active Granzyme B. Cells from previously infected animals had higher Granzyme B response than animals with no previous infection. In animals with no previous clinical infections, irradiated S. aureus antigen induced lower response than live S. aureus. Furthermore, bovine neutrophils generate greater levels of cytokines when stimulated with supernatants from S. aureus induced T cells. This data indicates that previous infection can heighten the ability of cytotoxic T cells to produce Granzyme B and increase neutrophil function. The ability to increase presence of protective memory immune cells and innate immune cell function in the mammary gland will provide new vaccine targets for S. aureus.

Identification of human endogenous retrovirus (HERV-K(HML-2))-specific mucosal CD4+ T cell responses in HIV-1-exposed, seronegative individuals (P6195)

Abstract The transcriptional silence of human endogenous retroviruses (HERV) can be disrupted in HIV-1 infection. We have reported that the strength of the HERV-specific CD8+ T cell response predicts lower HIV-1 viral load in untreated adults. To determine whether HERV-K immunity plays a role in inhibiting HIV-1 replication at a major site of transmission and CD4+ T cell depletion, we assessed these responses in gut-associated lymphoid tissue (GALT) from rectosigmoid biopsies and blood of HIV-1 exposed, seronegative (HESN) individuals (n=6) by flow cytometry. CMV pp65- and HIV-1 Gag-specific T cells were also measured, and all responses were compared with peripheral antigen-specific responses in uninfected, low-risk controls (n=11). With the exception of stronger CMV-specific CD8+ T cell responses in the HESN group (p=0.024), there were no significant differences between the magnitudes of peripheral antigen-specific T cell responses in the HESN vs. controls. However, HESN subjects had remarkably robust mucosal HIV-1- (median %CD4+cytokine+cells=2.67) and HERV-specific (median %CD4+cytokine+cells=3.41) CD4+ responses, which were much stronger than the corresponding PBMC responses (p=0.0079 and p&amp;lt;0.0001, respectively). These findings suggest that the GALT is an important site of HERV-K expression and immunity in individuals exposed to HIV-1, and should be further investigated in the context of novel vaccine strategies that target conserved antigens such as HERV.

Study of the PBMC response to TGN1412 and visilizumab in the “lymph node like” cytokine release assay format (P6327)

Abstract Recently, a new format for the cytokine release assay (CRA) used to predict the in vivo safety of therapeutic antibodies was developed. In this assay, normal donor PBMCs are pre-incubated at high density for two days to make the cells more “lymph node like” (LNL) and then are incubated with soluble antibody. The assay was developed in response to the failure of previous in vitro stimulation formats to predict the strong cytokine storm shown in humans by TGN1412, a CD28 superagonist human IgG4 antibody. Similar to TGN1412, previous formats failed to predict the strong cytokine response elicited in human patients by visilizumab, a human IgG2a anti-CD3 specific antibody. Here we compare and contrast the PBMC response in the LNL format to TGN1412 and visilizumab. Activation of PBMC subset populations, cytokine profile and cellular source of cytokines were analyzed. For the majority of donors tested, the LNL format of CRA detected PBMC cytokine responses to both TGN1412 and visilizumab, which were consistent with those cytokine responses previoulsly observed in vivo. Our study of two human IgG antibodies targeting different T cell activating receptors suggests that the LNL format of CRA may be a valuable predictor of in vivo cytokine release by T lymphocyte specific biologics.

Peripheral Monocyte Expression of the Chemokine Receptors CCR2, CCR5 and CXCR3 is Altered at Parturition in Healthy Women and in Women with Systemic Lupus Erythematosus

Monocytes are precursors of macrophages and recruited to the uterus throughout pregnancy to perform important immunological functions. In this study, we hypothesized that pregnant women have reduced peripheral monocyte expression of chemokine receptors and alterations in PBMC responses to microbial stimuli as an adaption to pregnancy and that these changes are less pronounced in women with autoimmunity. We therefore investigated the chemokine receptor expression, migratory behaviour and responses to microbial stimulation of peripheral monocytes from pregnant women at parturition (n = 13) and from non-pregnant women (n = 9). In addition, we compared healthy pregnant women with women suffering from SLE (n = 5), a condition with pronounced systemic inflammation increasing the risk for pregnancy complications. We demonstrate that peripheral monocytes are affected by pregnancy with reduced percentages of CCR2+, CCR5+ and CXCR3+ monocytes of both classical (CD16-) and inflammatory (CD16+) subsets and that the trophoblast-secreted chemokine CCL2/MCP-1 recruited monocytes of both subsets in vitro. Further, PBMCs from pregnant women had a divergent response to microbial stimulation with lower CCL5/RANTES and higher CCL2/MCP-1 secretion compared with non-pregnant women. In addition, pregnant women had lower basal PBMC-secretion of CCL5/RANTES and higher basal secretion of IL-10 and CCL2/MCP-1. Interestingly, the women with SLE responded similar to pregnancy as did healthy women with lower percentages of CCR2+, CCR5+ and CXCR3+ monocytes. However, they had increased expression of CCR5 on CD16+ monocytes and heightened PBMC-secretion of CCL5/RANTES. In conclusion, our data indicate that monocyte chemokine receptor expression and the chemokine milieu during pregnancy are tightly regulated to support pregnancy.

An Unbiased Genome-Wide Mycobacterium tuberculosis Gene Expression Approach To Discover Antigens Targeted by Human T Cells Expressed during Pulmonary Infection

Mycobacterium tuberculosis is responsible for almost 2 million deaths annually. Mycobacterium bovis bacillus Calmette-Guérin, the only vaccine available against tuberculosis (TB), induces highly variable protection against TB, and better TB vaccines are urgently needed. A prerequisite for candidate vaccine Ags is that they are immunogenic and expressed by M. tuberculosis during infection of the primary target organ, that is, the lungs of susceptible individuals. In search of new TB vaccine candidate Ags, we have used a genome-wide, unbiased Ag discovery approach to investigate the in vivo expression of 2170 M. tuberculosis genes during M. tuberculosis infection in the lungs of mice. Four genetically related but distinct mouse strains were studied, representing a spectrum of TB susceptibility controlled by the supersusceptibility to TB 1 locus. We used stringent selection approaches to select in vivo-expressed M. tuberculosis (IVE-TB) genes and analyzed their expression patterns in distinct disease phenotypes such as necrosis and granuloma formation. To study the vaccine potential of these proteins, we analyzed their immunogenicity. Several M. tuberculosis proteins were recognized by immune cells from tuberculin skin test-positive, ESAT6/CFP10-responsive individuals, indicating that these Ags are presented during natural M. tuberculosis infection. Furthermore, TB patients also showed responses toward IVE-TB Ags, albeit lower than tuberculin skin test-positive, ESAT6/CFP10-responsive individuals. Finally, IVE-TB Ags induced strong IFN-γ(+)/TNF-α(+) CD8(+) and TNF-α(+)/IL-2(+) CD154(+)/CD4(+) T cell responses in PBMC from long-term latently M. tuberculosis-infected individuals. In conclusion, these IVE-TB Ags are expressed during pulmonary infection in vivo, are immunogenic, induce strong T cell responses in long-term latently M. tuberculosis-infected individuals, and may therefore represent attractive Ags for new TB vaccines.

Chlamydia Pneumoniae (Cpn) - Induced in Vitro Interferon Gamma (IFN-γ) and Interleukin 2 (IL-2) Responses of PBMC From Asthmatics

Chlamydia Pneumoniae (Cpn) - Induced in Vitro Interferon Gamma (IFN-γ) and Interleukin 2 (IL-2) Responses of PBMC From Asthmatics

TLR ligands of ryegrass pollen microbial contaminants enhance Th1 and Th2 responses and decrease induction of Foxp3hi regulatory T cells

Microbial contamination of grass pollens could affect sensitization, subsequent allergic response, and efficacy of allergen-specific immunotherapy. We investigated whether bacterial immunomodulatory substances can direct PBMC responses of allergic and nonatopic subjects against ryegrass pollen (RGP) toward Th1, Th2, or regulatory T (Treg) cells. Aqueous extracts of RGP with high or low LPS were fractionated into large and small molecular weight (MW) components by diafiltration. CFSE-labeled PBMCs from allergic and nonatopic subjects were stimulated with RGP extracts (RGPEs) and analyzed for cytokine secretion and T-cell responses. High LPS RGPE increased IFN-γ(+) Th1 and IL-4(+) Th2 effector cell induction and consistently decreased CD4(+) Foxp3(hi) Treg-cell induction. IL-10-producing T-cell frequency was unaltered, but IL-10 secretion was increased by high LPS RGPE. RGPE-stimulation of TLR-transfected cell lines revealed that high LPS pollen also contained a TLR2-ligand, and both batches a TLR9-ligand. Beta-1,3-glucans were detected in large and small MW fractions and were also T-cell stimulatory. In conclusion, coexposure to allergen and proinflammatory microbial stimuli does not convert an established Th2- into a Th1-response. Instead, proinflammatory responses are exacerbated and Foxp3(hi) Treg-cell induction is decreased. These findings show that adjuvants for specific immunotherapy should enhance Treg cells rather than target immune deviation from Th2 to Th1.

Interferon-Gamma Production to Non-Polymorphic HLA Class 2 Derived Peptides Correlates with Those to HLA Class 1 Derived Peptides in Long-Term Renal Transplant Patients

Introduction: In long-term renal transplant patients we have shown a response to non-polymorphic peptides derived from the HLA Class 1 α3-domain: cryptic self-epitopes (AJT 2007). These responses were more common in patients with evidence of deteriorating graft function suggesting that measures of immune function based on responses to these peptides may be used as a basis for an assay of T cell responses to HLA that can be standardized across the population. We hypothesized that long-term renal transplant recipients would also respond to non-polymorphic HLA class 2 derived peptides and these responses would provide an opportunity to refine an assay that could be tested prospectively. Methods: An ‘in silico’ approach was used to design potential peptide epitopes (13-19 amino acids long) derived from the distal β1 and β2 domain of HLA-DR (sequences with significant homology to other HLA class 2 molecules). This used an EpiMatrix algorithm for epitope prediction and calculated the grand average of hydropathicity index to exclude peptides that were extremely insoluble. In contrast to our approach with class 1 peptides we did not then test for binding of these peptides to class 2 molecules in vitro but used them directly to test PBMC responses in transplant recipients. Peptides were synthesized using Fmoc Chemistry (GL Biochem, Shanghai, China). Recruits were at least 18 months post transplantation, recruited as previously described; interferon ▪ ELISPOT was performed and positive responses defined as previously described. The responses to class 2 peptides were compared to those previously documented against single or multiple class 1 peptides (Transplantation 2011). Results: The mean values for responses to HLA class 1 and class 2 peptides in healthy controls was 2.86 ± 0.35 (n=23) and 2.30 ± 0.13 (n=19) respectively but for transplant patients was 5.54 ± 0.288 (p< 10-3) and 4.45 ± 0.19 (p=0.02) respectively. The response to HLA class 2 derived peptides correlated with responses to class 1 derived peptides, tested singly or as mixes (p< 10-4). The numbers discordant between the two groups however, preclude any meaningful interpretation of clinical correlates of differential responses.Table: [Reponses to HLA Class 1 & Class 2 Derived Peptides]Conclusion: These data demonstrate a significant correlation between responses to non-polymorphic HLA class 1 and class 2 peptides. They imply that indirect alloresponses most frequently arise to both class 1 and class 2 peptides, although it cannot be proved that these particular responses are pathogenic. They suggest that a limited peptide set is likely to be sufficient to identify patients who response to such peptides; the clinical significance of which requires testing in a prospective manner.

Non-tuberculous mycobacteria and their surface lipids efficiently induced IL-17 production in human T cells

Interleukin-17 (IL-17) is produced by a subset of CD4+ T helper (Th) lymphocytes known as Th17 cells. In humans, IL-1β, enhanced by IL-6 and IL-23 is crucial for differentiation of these cells. IL-17 evokes inflammation and is involved in host defence against microorganisms, although little is known about its role in diseases caused by non-tuberculous mycobacteria. The genus Mycobacterium contains both obligate and opportunistic pathogens as well as saprophytes, and the mycobacterial cell envelope is unique in its abundance of lipids. Here we investigated IL-17 and IL-23 production in human PBMC in response to intact UV-inactivated mycobacteria and mycobacterial surface lipids from two opportunistic (Mycobacterium avium and Mycobacterium abscessus) and one generally non-pathogenic (Mycobacterium gordonae) species. Representative Gram-positive (Enterococcus faecalis, Streptococcus mitis) and Gram-negative (Escherichia coli) bacteria were included as controls. Intact mycobacteria induced production of large amounts of IL-17, while IL-17 responses to control bacteria were negligible. Purified CD4+ T cells, but not CD4-depleted cell fractions, produced this IL-17. Isolated mycobacterial surface lipids induced IL-17, but not IL-23 production. The ability of the non-tuberculous mycobacteria to induce IL-17 production in CD4+ T cells was the same regardless of the pathogenic potential of the particular mycobacterial species.

Analysis of T Cell Responses to the Major Allergens from German Cockroach: Epitope Specificity and Relationship to IgE Production

Bla g allergens are major targets of IgE responses associated with cockroach allergies. However, little is known about corresponding T cell responses, despite their potential involvement in immunopathology and the clinical efficacy of specific immunotherapy. Bioinformatic predictions of the capacity of Bla g 1, 2, 4, 5, 6, and 7 peptides to bind HLA-DR, -DP, and -DQ molecules, and PBMC responses from 30 allergic donors, identified 25 T cell epitopes. Five immunodominant epitopes accounted for more than half of the response. Bla g 5, the most dominant allergen, accounted for 65% of the response, and Bla g 6 accounted for 20%. Bla g 5 induced both IL-5 and IFN-γ responses, whereas Bla g 6 induced mostly IL-5, and, conversely, Bla g 2 induced only IFN-γ. Thus, responses to allergens within a source are independently regulated, suggesting a critical role for the allergen itself, and not extraneous stimulation from other allergens or copresented immunomodulators. In comparing Ab with T cell responses for several donor/allergen combinations, we detected IgE titers in the absence of detectable T cell responses, suggesting that unlinked T cell-B cell help might support development of IgE responses. Finally, specific immunotherapy resulted in IL-5 down modulation, which was not associated with development of IFN-γ or IL-10 responses to any of the Bla g-derived peptides. In summary, the characteristics of T cell responses to Bla g allergens appear uncorrelated with IgE responses. Monitoring these responses may therefore yield important information relevant to understanding cockroach allergies and their treatment.

Phase I trial of extended-dose anti-PD-1 antibody BMS-936558 with a multipeptide vaccine for previously treated stage IV melanoma.

8582 Background: BMS-936558, a fully human IgG4 anti-PD-1 antibody, has shown clinical activity in melanoma and renal cancer. Methods: 30 HLA A*0201 positive patients with unresectable, previously treated stage IV melanoma received MART-1/gp100/NY-ESO-1 peptides with adjuvant Montanide ISA 51 injected subcutaneously with BMS-936558 at 1, 3 or 10 mg/kg intravenously every 2 weeks for 24 weeks, followed by BMS-936558 alone every 3 months. No patient had prior ipilimumab. Endpoints were toxicity, correlative studies of immunity measured by ELISPOT and T-cell phenotype assays, and response by RECIST. Results: Median age was 62, with 14 men and 16 women. 73% of patients had M1c disease. One patient had dose limiting optic neuritis; one patient had dose limiting grade 3 interstitial pneumonitis. Of 10 patients at 1 mg/kg, 2 had confirmed partial responses (PR), both one year from initiation of study. 13 patients were treated at the 3 mg/kg cohort; 3 patients did not complete more than 3 doses secondary to progressive disease and one withdrew consent. Of 12 evaluable patients, 3 had confirmed PRs and 2 unconfirmed PRs. In the 10 mg/kg cohort, thus far 6 patients completed one cycle with 2 unconfirmed PRs and one patient stable. Only 2 of the 9 responders after one cycle have progressed with a median follow-up of 7 months. 2/6 patients failing BMS-936558 responded to ipilimumab. At week 12, patients in all cohorts had decreased PD-1 on CD4 and CD8 T-cells (p&lt;0.0001), increased CD4 CTLA-4 levels (p=0.01) and dose related decreases in CD8 T-cells whereas CD4 T-cells increased (both p&lt;0.05). ELISPOT assays showed that patients at all doses had no increased responses in fresh PBMC to MART-1/gp100, but showed dose related decreased responses to viral (CMV, EBV, FLU) peptides over time (p=0.04). Conclusions: BMS-936558 in combination with a peptide vaccine is well tolerated at 1 and 3mg/kg. Accural to the 10mg/kg cohort is on-going. Responses have been observed at all dose levels. PD-1 levels on T-cells decreased, and CTLA-4 levels increased in CD4 T cells in all cohorts. There was a dose-related decrease in CD8 T-cells and in viral-specific T-cells. Further study of BM-936558 is warranted to determine its optimal dose.

FcGR2A genotype and change in gene expression of effecter cells in peripheral blood after administration of trastuzumab (T).

e11065 Background: We have previously reported that FcGR2A polymorphisms were associated with clinical outcome of trastuzumab (T) in both neoadjuvant and metastatic settings in patients (pts) with HER2-positive breast cancer (Tamura et al, Ann Oncol 2011). We explored if antibody-dependent cell-mediated cytotoxicity (ADCC) can be observed as a change in gene expression of PBMC (effecter cells of ADCC) in patients receiving T according to FcGR2A phenotypes. Methods: FcGR2A genotypes in 34 patients with HER2-positive metastatic breast cancer were determined in the previously reported prospective study (Tamura et al, Ann Oncol 2011). All pts received single agent T (Starting dose 4mg/kg, maintenance dose 2mg/kg) as the first-line treatment for metastatic breast cancer. PBMC was purified from peripheral blood collected before T, at 1 wk and 8 wk of weekly T. Total RNA was extracted from PBMC and gene expressions were measured using Affimetrix GeneChip Human Genome U133 Plus 2.0 array. Gene set enrichment analysis (GSEA) ( www.broadinstitute.org/gsea ) using KEGG pathway DB was carried out to explore differentially expressed gene sets (GSs) after 1wk and 8 wk administration of T compared to baseline. GSs with significance level of p&lt;0.05 and FDA&lt;0.25 were considered statistically significant. Results: There were 15 and 19 patients with FcG2A- 131 HH and HR/RR genotypes, respectively. There were no significant GSs differentially expressed at 1 wk compared to baseline in both HH and HR/RR cohorts. 11 GSs were differentially expressed at 8 wk in HH cohort, whereas there were no significant GSs differentially expressed at 8wk in HR/RR cohort. The upregulated GSs at 8 wk in HH cohort included KEGG_NATURAL_KILLER_CELL_MEDIATED_CYTOTOXICIY and KEGG_ANTIGEN_PROCESSING_AND_PRESENTATION. Conclusions: Differential pharmacogenomic response of PBMC to T according to FcG2A genotype supports the hypothesis that ADCC is associated with the mechanism of action of T.

Towards a xeno-free and fully chemically defined cryopreservation medium for maintaining viability, recovery, and antigen-specific functionality of PBMC during long-term storage

Analysis of cryopreserved peripheral mononuclear cells (PBMC) is important for evaluating new vaccines in immune based therapies and in pathogenesis studies. To ensure comparable assay results from different laboratories and points of time, collaborative research in multicenter trials needs reliable and reproducible cryopreservation protocols that maintain cell viability and functionality. Current cryomedia consist largely of fetal bovine serum (FBS), a natural mix of growth factors, cytokines, and undefined compounds. Standardized procedures are not possible, as FBS can affect the antigen-specific T-cell response, the most important parameter in functionality assays. Also, worldwide sample exchange is complicated by the strict import restrictions on FBS, because of transfection risk.After establishing a serum-free cryopreservation protocol that maintains cell viability, recovery and antigen-specific T-cell response of PBMC comparably to FBS-based cryomedia (Germann et al., 2011), the aim of this study was the complete avoidance of animal proteins and products in combination with efficient cryopreservation.As long-term stability of the cryopreservation process is crucial for retrospective evaluation of samples at different points of time, PBMC were analyzed after storage for maximal four weeks and again after approximately six months.The cryopreservation efficiency of the protein-free and fully chemically defined cryomedium was comparable to FBS-medium after storage for few weeks and several months. Directly after thawing, this medium yielded viabilities over 97% and recovery values over 84%. Also, the specific T-cell functionality was preserved. Additionally, short-term and six month cryopreservation gave comparable results. The fully chemically defined medium presented here will increase standardization and reproducibility of analysis in multicenter-studies or in retrospective evaluation.

The role of IL-21 in rescuing CD8 T Cell responses in chronically infected HIV patients (105.38)

Abstract CD4 T cells are vital in regulating CD8 T cell responses during chronic viral infections. Notably, the hallmark of HIV infection is the severe loss of CD4 T cells resulting in defective CD8 T cell responses. One consequence of CD4 depletion following HIV infection is the loss of interleukin-21 (IL-21) production by the CD4 T cells, which has been demonstrated to be a critical factor in promoting viral control in murine studies. However, it is unknown whether IL-21 can rescue the exhausted CD8 T cells in chronically infected HIV+ patients. In this study we build upon novel murine studies to demonstrate a key role for IL-21 in improving CD8 T cell responses in PBMC from chronically infected HIV patients. To determine whether IL-21 can rescue exhausted CD8 T cells, PBMCs from HIV patients with viral loads &amp;gt; 10,000 RNA copies/mL were incubated with IL-21. Following re-stimulation with overlapping Gag-peptide, IL-21 enhanced the production of IFN-γ by CD8 T cells although no effect was observed on IL-2 or TNF-α production. In addition, an increase in the central memory (CD45RO+CD27+) population was observed with IL-21 culture along with higher MFI of Bcl-6 levels. Interestingly, IL-21 cultured CD8 T cells expressed higher MFI of PD-1 even though CD8 T cell function was enhanced. Significantly, these results provide an approach in which IL-21 can be potentially used to enhance CD8 T cell responses in chronically infected HIV patients, whose CD4 counts are low or defective.