Standardization of cryopreserved peripheral blood mononuclear cells through a resting process for clinical immunomonitoring--Development of an algorithm.

Flow cytometry, as a powerful tool for immunomonitoring and quality control of peripheral blood mononuclear cells (PBMCs), is routinely used in clinical studies. However, flow cytometry based assays for cryopreserved peripheral blood mononuclear cells (cPBMCs) constitute a challenge. Down-regulation of surface and intracellular markers, as well as impairment of cell function might result from cryopreservation. Furthermore, protocols for resting cPBMCs are available but diverse. Therefore, we performed a standardization of the resting process concerning resting position, cell concentration, resting period and material of cell culture tubes as well as culture media. We further investigated the influence of resting on the phenotype and functionality of T cells comparing fresh PBMCs as gold standard to rested and non-rested cPBMCs. Polychromatic flow cytometry staining, peptide-MHC class I restricted tetramer staining and intracellular cytokine staining as major methods were used. Our results revealed that a horizontal position, a cell concentration of 2 to 5 × 10(6) cells/ml and an overnight resting phase is beneficial to eliminate dead or dying cells in cPBMCs with a mean cell loss of 14% overall cell populations. In addition, the quality and quantity of regulatory T cells and antigen specific T cells recovered upon resting. For multifunctional T cells a decrease of activation threshold in the way of a twofold mean fluorescence intensity (MFI) and increase of degranulation marker CD107a, co-stimulatory marker CD28, adhesion molecule CD62L as well as the ability to secrete antiviral cytokines like interferon gamma (IFN-γ), tumor necrosis factor (TNF), and interleukin 2 (IL-2) comparable to fresh PBMCs were observed. However, based upon our data resting is not helpful for the flow cytometric analyses of myeloid-derived suppressor cells (MDSCs) and large/intermediate size lymphocytes which rather decreased/vanished ex vivo. Therefore, we developed an algorithm to indicate for which cell population and for which type of analyses the resting process is useful or not.

Human T cells expressing two additional receptors (TETARs) specific for HIV-1 recognize both epitopes.

Adoptive TCR transfer against rapidly mutating targets, such as HIV-1 or cancer, must counteract corresponding immune escape. Hence, we generated T cells expressing two additional receptors (TETARs) specific for HIV-1 by TCR mRNA electroporation. An HLA-A2-restricted gag-specific TCR and an HLA-B13-restricted nef-specific TCR were chosen. When both TCRs were transfected simultaneously, strong competitive effects occurred that were overcome by replacing the human constant domains of one TCR with murine counterparts and adapting the amounts of TCR-RNA used for transfection. The resulting TETAR responded to both epitopes with cytokine secretion and cytotoxic function. Cell sorting revealed that one individual cell indeed recognized both epitopes. The T cells diminished their reactivity to each epitope after stimulation but sequentially killed targets that presented the gag epitope and then targets that presented the nef epitope, or vice versa. Taken together, TETARs represent a sophisticated tool to study TCR functionality and might be a useful strategy in immunotherapy.

Definition and characterization of novel HLA-*A02-restricted CD8+ T cell epitopes derived from JCV polyomavirus with clinical relevance.

Human JC and BK polyomaviruses (JCV/BKV) can establish a latent infection without any clinical symptoms in healthy individuals. In immunocompromised hosts infection or reactivation of JCV and BKV can cause lethal progressive multifocal leukoencephalopathy (PML) and hemorrhagic cystitis, respectively. Vaccination with JCV/BKV derived antigen epitope peptides or adoptive transfer of virus-specific T cells would constitute an elegant approach to clear virus-infected cells. Furthermore, donor leukocyte infusion (DLI) is another therapeutic approach which could be helpful for patients with JCV/BKV infections.So far, only few immunodominant T cell epitopes of JCV and BKV have been described and therefore is a fervent need for the definition of novel epitopes. In this study, we identified novel T cell epitopes by screening libraries of overlapping peptides derived from the major capsid protein VP1 of JCV. Virus like particles (VLPs) were used to confirm naturally processing. Two human leucocyte antigen (HLA)-A*02-restricted epitopes were characterized by fine mapping with overlapping peptides and nonamer peptide sequences were identified. Cytokine release profile of the epitope-specific T cells was analyzed by enzyme-linked immunospot (ELISPOT) assays and by flow cytometry. We demonstrated that T cell responses were of polyfunctional nature with the potential of epitope-specific killing and cross-reactivity between JCV and BKV. These novel epitopes might constitute a new potential tool to design effective diagnostic and therapeutic approaches against both polyomaviruses.

Frequency of expression and generation of T-cell responses against antigens on multiple myeloma cells in patients included in the GMMG-MM5 trial.

BACKGROUND: Raising T-cell response against antigens either expressed on normal and malignant plasma cells (e.g. HM1.24) or aberrantly on myeloma cells only (e.g. cancer testis antigens, CTA) by vaccination is a potential treatment approach for multiple myeloma. RESULTS: Expression by GEP is found for HM1.24 in all, HMMR in 318/458 (69.4%), MAGE-A3 in 209/458 (45.6%), NY-ESO-1/2 in 40/458 (8.7%), and WT-1 in 4/458 (0.8%) of samples with the pattern being confirmed by RNA-sequencing. T-cell-activation is found in 9/26 (34.6%) of patient samples, i.e. against HM1.24 (4/24), RHAMM-R3 (3/26), RHAMM1-8 (2/14), WT-1 (1/11), NY-ESO-1/2 (1/9), and MAGE-A3 (2/8). In 7/19 T-cell activation responses, myeloma cells lack respective antigen-expression. Expression of MAGE-A3, HMMR and NY-ESO-1/2 is associated with adverse survival. EXPERIMENTAL DESIGN: We assessed expression of HM1.24 and the CTAs MAGE-A3, NY-ESO-1/2, WT-1 and HMMR in CD138-purified myeloma cell samples of previously untreated myeloma patients in the GMMG-MM5 multicenter-trial by gene expression profiling (GEP; n = 458) and RNA-sequencing (n = 152) as potential population regarding vaccination trials. We then validated the feasibility to generate T-cell responses (n = 72) against these antigens by IFN-γ EliSpot-assay (n = 26) related to antigen expression (n = 22). Lastly, we assessed survival impact of antigen expression in an independent cohort of 247 patients treated by high-dose therapy and autologous stem cell transplantation. CONCLUSIONS: As T-cell responses can only be raised in a subfraction of patients despite antigen expression, and the number of responses increases with more antigens used, vaccination strategies should assess patients' antigen expression and use a "cocktail" of peptide vaccines.

Differences in Expansion Potential of Naive Chimeric Antigen Receptor T Cells from Healthy Donors and Untreated Chronic Lymphocytic Leukemia Patients.

INTRODUCTION: Therapy with chimeric antigen receptor T (CART) cells for hematological malignancies has shown promising results. Effectiveness of CART cells may depend on the ratio of naive (TN) vs. effector (TE) T cells, TN cells being responsible for an enduring antitumor activity through maturation. Therefore, we investigated factors influencing the TN/TE ratio of CART cells. MATERIALS AND METHODS: CART cells were generated upon transduction of peripheral blood mononuclear cells with a CD19.CAR-CD28-CD137zeta third generation retroviral vector under two different stimulating culture conditions: anti-CD3/anti-CD28 antibodies adding either interleukin (IL)-7/IL-15 or IL-2. CART cells were maintained in culture for 20 days. We evaluated 24 healthy donors (HDs) and 11 patients with chronic lymphocytic leukemia (CLL) for the composition of cell subsets and produced CART cells. Phenotype and functionality were tested using flow cytometry and chromium release assays. RESULTS: IL-7/IL-15 preferentially induced differentiation into TN, stem cell memory (TSCM: naive CD27+ CD95+), CD4+ and CXCR3+ CART cells, while IL-2 increased effector memory (TEM), CD56+ and CD4+ T regulatory (TReg) CART cells. The net amplification of different CART subpopulations derived from HDs and untreated CLL patients was compared. Particularly the expansion of CD4+ CARTN cells differed significantly between the two groups. For HDs, this subtype expanded >60-fold, whereas CD4+ CARTN cells of untreated CLL patients expanded less than 10-fold. Expression of exhaustion marker programmed cell death 1 on CARTN cells on day 10 of culture was significantly higher in patient samples compared to HD samples. As the percentage of malignant B cells was expectedly higher within patient samples, an excessive amount of B cells during culture could account for the reduced expansion potential of CARTN cells in untreated CLL patients. Final TN/TE ratio stayed <0.3 despite stimulation condition for patients, whereas this ratio was >2 in samples from HDs stimulated with IL-7/IL-15, thus demonstrating efficient CARTN expansion. CONCLUSION: Untreated CLL patients might constitute a challenge for long-lasting CART effects in vivo since only a low number of TN among the CART product could be generated. Depletion of malignant B cells before starting CART production might be considered to increase the TN/TE ratio within the CART product.

Peptide vaccination in the presence of adjuvants in patients after hematopoietic stem cell transplantation with CD4+ T cell reconstitution elicits consistent CD8+ T cell responses.

Rationale: Patients receiving an allogeneic stem cell graft from cytomegalovirus (CMV) seronegative donors are particularly prone to CMV reactivation with a high risk of disease and mortality. Therefore we developed and manufactured a novel vaccine and initiated a clinical phase I trial with a CMV phosphoprotein 65 (CMVpp65)-derived peptide. Methods: Ten patients after allogeneic stem cell transplantation received four vaccinations at a biweekly interval. All patients were monitored for CMVpp65 antigenemia. Flow cytometry for CMV-specific CD8+ and γδ T cells as well as neutralizing anti-CMV antibodies were correlated to clinical parameters. Results: The vaccination was well tolerated. Seven of nine patients cleared CMVpp65 antigenemia after four vaccinations and are still free from antigenemia to this day. Two patients with CMV reactivation showed persisting CMV antigenemia. One patient received prophylactic vaccination and did not develop antigenemia. An increase of up to six-fold in frequency of both CMV-specific CD8+ T cells and/or Vδ2negative γδ T cells was detected. Titers of neutralizing antibodies increased up to the tenfold. Humoral and cellular immune responses correlated with clearance of CMV. Conclusion: In summary, CMVpp65 peptide vaccination for patients after allogeneic stem cell transplantation at high risk for CMV reactivation was safe, well tolerated and clinically encouraging. A study in solid-organ transplant patients is ongoing.

T-cell receptor transfer for boosting HIV-1-specific T-cell immunity in HIV-1-infected patients.

OBJECTIVES: Strategies to cure HIV-1 infection require the eradication of viral reservoirs. An innovative approach for boosting the cytotoxic T-lymphocyte response is the transfer of T-cell receptors (TCRs). Previously, we have shown that electroporation of TCR-encoding mRNA is able to reprogram CD8 T cells derived from healthy donors. So far, it is unknown whether the transfer of HIV-1-specific TCRs is capable to reprogram CD8 T cells of HIV-1-infected patients. To assess the efficiency of TCR-transfer by mRNA electroporation and the functionality of reprogramed T cells in HIV-1-infected patients, we performed an in-vitro analysis of TCR-transfer into T cells from HIV-1-infected patients in various stages of disease and from healthy controls. METHODS: Peripheral blood mononuclear cells from 16 HIV-1-infected patients (nine HLA-A02-positive, seven HLA-A02-negative) and from five healthy controls were electroporated with mRNA-constructs encoding TCRs specific for the HLA-A02/HIV-1-gag p17 epitope SLYNTVATL (SL9). Functionality of the TCRs was measured by γIFN-ELISpot assays. RESULTS: SL9/TCR transfection into peripheral blood mononuclear cells from both HLA-A02-positive and HLA-A02-negative HIV-1-infected patients and from healthy blood donors reprogramed T cells for recognition of SL9-presenting HLA-A02-positive cells in γIFN-ELISpot assays. SL9/TCR-transfer into T cells from an immunodeficient AIDS patient could induce recognition of SL9-expressing target cells only after reversion of T-cell dysfunction by antiretroviral therapy. CONCLUSION: The transfer of HIV-1-p17-specific TCRs into T cells is functional both in HIV-1-infected patients as well as in healthy blood donors. TCR-transfer is a promising method to boost the immune system against HIV-1.

Chimeric Antigen Receptor T Cell Therapy Targeting CD19-Positive Leukemia and Lymphoma in the Context of Stem Cell Transplantation.

Novel therapies with chimeric antigen receptor (CAR)-transduced T cells (TCs) sparked new hope for patients with relapsed or refractory CD19-positive leukemia or lymphoma even after stem cell therapies. This review focuses on CARs recognizing the B cell antigen CD19. Both retroviral and lentiviral vectors are used, encoding various anti-CD19 CAR constructs comprising costimulatory molecules such as CD28, CD137/4-1BB, and OX40 either alone (second-generation CARs) or in combination (third-generation CARs). Current, up-to-date published studies on anti-CD19 CAR therapy for acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), and non-Hodgkin lymphoma (NHL) with observed side effects are discussed and an outlook on 58 ongoing trials is given. Clinical responses were achieved in up to 81% of ALL, 50% of CLL, and 40% of NHL patients. Factors with potential influence on the clinical outcome might be the design of the vector, the preconditioning regimen, and the number and quality of transfused CAR TCs. The applicability of clinical CAR TC therapy might include relapse after allogeneic stem cell transplantation (alloSCT), and ineligibility for or "bridging" until alloSCT. In summary, CAR therapy represents a highly promising treatment option even in heavily pretreated patients.

Cross-Reactivity Between Influenza Matrix- and HIV-1 P17-Specific CTL-A Large Cohort Study.

BACKGROUND: It has been reported that HIV-1-specific cytotoxic T cells (CTL) recognizing the HLA-A2-restricted p17 epitope SLYNTVATL (SL9) can cross-react with the HLA-A2-restricted influenza matrix epitope GILGFVFTL (GL9). So far, the prevalence of GL9-cross-reacting HIV-1-specific CTL in larger cohorts of HIV-1-infected patients is unknown, and there are no data yet on whether SL9/GL9-cross-reactive CTL may influence the course of HIV-1 infection. METHODS: We analyzed the presence of SL9/GL9-cross-reacting CTL in a cohort of 175 HLA-A2-positive HIV-1-infected patients. Peripheral blood mononuclear cells were stimulated in vitro with SL9 and GL9 peptides, and outgrowing cell lines regarding cross-reactivity and recognition of viral variants in γ-interferon enzyme-linked immunospot assays were analyzed. RESULTS: SL9- and GL9-specific CTL could be generated in 52.6% and 53.7% of 175 patients, respectively. Both SL9- and GL9-specific CTL were more frequently observed in patients on antiretroviral therapy (ART). Of the 92 SL9-specific CTL and the 94 GL9-specific CTL, 65.2% and 66%, respectively, showed at least partial SL9/GL9 cross-reactivity. SL9/GL9-cross-reactive CTL could be detected in 42.9% of the 175 patients. Recognition of SL9 was associated with lower viral loads and higher CD4 cell counts in patients on ART. Patients with GL9/SL9 cross-reactivity displayed similar CD4 cell counts than patients without GL9/SL9-cross-reactive cells. GL9/SL9-cross-reactive cells were associated with higher viral loads in patients on ART. CONCLUSIONS: Partially SL9/GL9-cross-reactive CTL are frequently observed in HIV-1-infected patients. So far, we could not detect a significant influence of the presence of SL9/GL9-cross-reacting CTL on the course of HIV-1 infection.

HIV-1 mRNA electroporation of PBMC: a simple and efficient method to monitor T-cell responses against autologous HIV-1 in HIV-1-infected patients.

Efficient monitoring of HIV-1-specific T-cells is crucial for the development of HIV-1 vaccines and immunotherapies. Currently, mainly peptides and vaccinia vectors are used for detection of HIV-1-specific cytotoxic T-lymphocytes (CTL), however, as HIV-1 is a variable virus, it is unknown to what extent the T-cell response against the autologous virus is under- or overestimated by using antigens from heterologous viral strains. Therefore, we established a new method for immunomonitoring of CTL using electroporation of peripheral blood mononuclear cells (PBMC) with mRNA derived from autologous viral strains. From six HIV-1-infected patients virus derived mRNA was produced after PCR-based cloning of autologous gag (n=5) and/or nef genes (n=3) from plasma and electroporated into PBMC from patients and healthy donors. Electroporation of PBMC with mRNA resulted in efficient protein expression with good induction of γ-interferon (γ-IFN) release by specific T-cells comparable to peptide pools and better than recombinant vaccinia viruses. Three mRNA encoded autologous Gag proteins and one autologous mRNA encoded Nef protein were better recognized by autologous PBMC in comparison to heterologous mRNA encoded Gag or Nef proteins (SF2 or HXB2). However, in one case each, mRNA encoded autologous Gag or Nef, respectively, was recognized less efficiently due to the presence of CTL escape mutations. In summary, electroporation of PBMC with mRNA is a very efficient, easy and rapid method for immunomonitoring of HIV-1-specific T-cell responses against autologous viral strains. Our data demonstrate that patients' CTL responses against autologous viral strains may be under- or overestimated by using antigens from heterologous viral strains.

NONEXPRESSOR OF PATHOGENESIS-RELATED PROTEINS1 (NPR1) and some NPR1-related proteins are sensitive to salicylic acid.

NONEXPRESSOR OF PATHOGENESIS-RELATED PROTEINS1 (NPR1; also known as NIM1) is a master regulator of systemic acquired resistance (SAR). SAR is induced by salicylic acid (SA), leading to the expression of PATHOGENESIS-RELATED (PR) genes. Current evidence suggests that NPR1 is part of a transcription complex tethered to activation sequence-1 (as-1)-like cis-acting elements in PR-1 gene promoters through TGA transcription factors, and that SA-dependent PR-1 gene expression is regulated by NIM1-INTERACTING (NIMIN) proteins. In Arabidopsis, NPR1 is active only after SA induction. Regulation of Arabidopsis NPR1 activity has been proposed to comprise cysteine-156 (Cys-156), mediating SA-induced cytoplasmic oligomer-nuclear monomer exchange, and Cys-521 and Cys-529, mediating SA-dependent transcriptional activation. Tobacco NPR1 does not harbour these residues. To understand the function of tobacco NPR1, we analysed its biochemical capabilities in a heterologous system: yeast. Tobacco NPR1 differs from Arabidopsis NPR1 in its subcellular localization and its transactivation potential. Yet, both tobacco and Arabidopsis NPR1, as well as tobacco NIM1-like1, alter some of their biochemical activities in response to SA. Whereas the addition of SA to yeast growth medium induces transcriptional activity in tobacco NPR1, its interaction with NIMIN2-type proteins is suppressed. The effects of SA are specific, sensitive and occur coordinately. They are abolished completely by mutation of the arginine residue within the invariable penta-amino acid motif LENRV, as present in the nonfunctional Arabidopsis nim1-4 allele. Furthermore, NPR1 proteins with the LENRV domain coincidently harbour a broad and strongly conserved NIMIN1/NIMIN2 binding site. Our data suggest that NPR1 and some NPR1-like proteins are sensitive to the plant hormone SA, altering some of their biochemical capabilities to enable stimulus-dependent gene expression. The sensitivity of NPR1 proteins to SA, together with their differential interaction with diverse NIMIN proteins, seems a plausible molecular basis for the timely and coordinated activation of PR genes during SAR.