Radiation-induced alteration of apatite on the surface of Mars: first in situ observations with SuperCam Raman onboard Perseverance.

Planetary exploration relies considerably on mineral characterization to advance our understanding of the solar system, the planets and their evolution. Thus, we must understand past and present processes that can alter materials exposed on the surface, affecting space mission data. Here, we analyze the first dataset monitoring the evolution of a known mineral target in situ on the Martian surface, brought there as a SuperCam calibration target onboard the Perseverance rover. We used Raman spectroscopy to monitor the crystalline state of a synthetic apatite sample over the first 950 Martian days (sols) of the Mars2020 mission. We note significant variations in the Raman spectra acquired on this target, specifically a decrease in the relative contribution of the Raman signal to the total signal. These observations are consistent with the results of a UV-irradiation test performed in the laboratory under conditions mimicking ambient Martian conditions. We conclude that the observed evolution reflects an alteration of the material, specifically the creation of electronic defects, due to its exposure to the Martian environment and, in particular, UV irradiation. This ongoing process of alteration of the Martian surface needs to be taken into account for mineralogical space mission data analysis.

Long-term safety and efficacy of lentiviral hematopoietic stem/progenitor cell gene therapy for Wiskott-Aldrich syndrome.

Patients with Wiskott-Aldrich syndrome (WAS) lacking a human leukocyte antigen-matched donor may benefit from gene therapy through the provision of gene-corrected, autologous hematopoietic stem/progenitor cells. Here, we present comprehensive, long-term follow-up results (median follow-up, 7.6 years) (phase I/II trial no. NCT02333760 ) for eight patients with WAS having undergone phase I/II lentiviral vector-based gene therapy trials (nos. NCT01347346 and NCT01347242 ), with a focus on thrombocytopenia and autoimmunity. Primary outcomes of the long-term study were to establish clinical and biological safety, efficacy and tolerability by evaluating the incidence and type of serious adverse events and clinical status and biological parameters including lentiviral genomic integration sites in different cell subpopulations from 3 years to 15 years after gene therapy. Secondary outcomes included monitoring the need for additional treatment and T cell repertoire diversity. An interim analysis shows that the study meets the primary outcome criteria tested given that the gene-corrected cells engrafted stably, and no serious treatment-associated adverse events occurred. Overall, severe infections and eczema resolved. Autoimmune disorders and bleeding episodes were significantly less frequent, despite only partial correction of the platelet compartment. The results suggest that lentiviral gene therapy provides sustained clinical benefits for patients with WAS.

Resetting the immune response after autologous hematopoietic stem cell transplantation for autoimmune diseases.

Autologous hematopoietic stem cell transplantation (AHSCT) is currently investigated as treatment for severe and refractory autoimmune diseases, such as multiple sclerosis (MS), systemic sclerosis (SSc), Crohn's disease (CD) and systemic lupus erythematosus. Randomized clinical trials in MS, SSc and CD have shown the efficacy of AHSCT to promote control of disease activity and progression, when compared to conventional treatment. The use of high dose immunosuppressive conditioning is essential to eliminate the autoimmune repertoire, and the re-infusion of autologous hematopoietic stem cells avoids long-term leucopenia by reconstitution of both immune and hematological systems. Recent studies showed that AHSCT is able to deplete the autoimmune compartment and further promote the formation of a new auto-tolerant immune repertoire, reducing the inflammatory milieu and leading to long-term clinical remission without any complementary post-graft treatment. Deep knowledge about the mechanisms of action related to AHSCT-induced remission is required for the management of possible post-AHSCT relapse and improvement of clinical protocols. This paper will review the mechanisms enrolled in the immune response resetting promoted by AHSCT in patients with autoimmune diseases.

Oligoclonal expansions of mucosal T cells in Crohn's disease predominate in NKG2D-expressing CD4 T cells.

Crohn's disease (CD) is an inflammatory pathology of the mucosal intestine that results from uncontrolled immune response towards commensal microbes. Clonal expansions of T cells have been found in patients with CD suggesting an antigen-specific stimulation of pathogenic T cells. Here we show, using T-cell receptor repertoire analysis by real-time PCR, that oligoclonal expansions are found in both CD8+ and CD4+ T cells in the blood and intestinal mucosa of CD patients. The majority of CD4+ T-cell-expanded clones are CD4+NKG2D+ T cells. These clonal expansions were found in both inflamed and neighboring healthy tissue and were persisting during the course of the disease. The presence of these CD4+NKG2D+ T-cell clones at the macroscopically normal edge of the surgical resection might be predictive of inflammation relapse post surgery.

Thymus and immune reconstitution after allogeneic hematopoietic stem cell transplantation in humans: never say never again.

Assessment of the host immune status is becoming a key issue in allogeneic hematopoietic stem cell transplantation (allo-HSCT). In the long-term follow-up of these patients, severe post-transplant infections, relapse or secondary malignancies may be directly related to persistent immune defects. In allo-HSCT, T-cell differentiation of donor progenitors within the recipient thymus is required to generate naive recent T-cell emigrants (RTE). These cells account for a durable T-cell reconstitution, generating a diverse T-cell receptor (TCR) repertoire and robust response to infections. It is now possible to quantify the production of RTE by measuring thymic T-cell receptor excision circles or 'TREC' which are small circular DNA produced during the recombination of the genomic segments encoding the TCR alpha chain. Here we discuss the role of thymic function in allo-HSCT. The pre-transplant recipient thymic function correlates with clinical outcome in terms of survival and occurrence of severe infections. Post-transplant, TREC analysis showed that the thymus is a sensitive target to the allogeneic acute graft-versus-host disease (GvHD) reaction but is also prone to recovery in young adult patients. In all, thymus is a key player for the quality of immune reconstitution and clinical outcome after allo-HSCT. Thymic tissue is plastic and it is a future challenge to halt or reverse thymic GVHD therapeutically by acting at the level of T-cell progenitors generation, thymic homing and/or epithelial thymic tissue preservation.

Outcomes, infections, and immune reconstitution after double cord blood transplantation in patients with high-risk hematological diseases.

Double unrelated cord blood transplant (dUCBT) has been used to circumvent cell dose limitation of single UCBT; however, few data are available describing outcomes, infectious disease, and immune recovery. We analyzed 35 consecutive dUCBT recipients with high-risk malignant disorders (n=21) and bone marrow failure syndromes (n=14). Median follow-up was 32 months. Conditioning regimen was myeloablative in 14 and reduced intensity in 21 patients. Median infused nucleated cell dose was 4 × 10(7) /kg. Median time to absolute neutrophil count >0.5 × 10(9) /L was 25 days. Cumulative incidence (CI) of acute grade II-IV graft-versus-host disease was 47%. Estimated overall survival at 2 years was 48%. CI of first viral infections at 1 year was 92%. We observed 49 viral infections in 30 patients, 34 bacterial infections in 19 patients, and 16 fungal or parasitic infections in 12 patients. Lymphocyte subset analyses were performed at 3, 6, 9, and >12 months after dUCBT. Decreased T-cell and B-cell counts with expansion of natural killer cells were observed until 9 months post transplantation. Recovery of thymopoiesis measured by T-cell receptor excision circles was impaired until 9 months after dUCBT, when the appearance of new thymic precursors was observed. Delayed immune recovery and high incidence of infectious complications were observed after dUCBT in patients with high-risk hematological diseases.

New tools in assessing immune reconstitution after hematopoietic stem cell transplantation.

BACKGROUND AND OBJECTIVES: Immune functions are impaired after allogeneic stem cell transplantation for several months depending on the age of the recipient, initial pathology, degree of HLA and minor histocompatibility antigens mismatches, origin and manipulation of the graft (unmanipulated or T-cell depleted bone marrow transplantation, cord blood) and post-transplantation events (acute or chronic graft-versus-host disease, relapse and infectious complications). MATERIAL AND METHODS, RESULTS AND CONCLUSION: In addition to lymphocyte phenotyping and functional assays, new tools are now available to monitor specific aspects of the immune response in the follow-up of hematopoietic stem cell transplantation: reconstitution of T cell diversity (spectratyping or Immunoscope), thymic function (TREC or "T-cell receptor rearrangement excision DNA circles") and antigen-specific T cell responses (HLA tetramers).

Molecular remission of chronic myeloid leukaemia following a non-myeloablative allogeneic peripheral blood stem cell transplant: in vivo and in vitro evidence for a graft-versus-leukaemia effect.

Two patients with chronic myeloid leukaemia (CML) received a non-myeloablative preparative regimen of cyclophosphamide and fludarabine, followed by an unmanipulated, G-CSF-mobilized, peripheral blood stem cell transplant from an HLA-identical sibling. Chimaerism, evaluated in myeloid and T-lymphoid lineages by PCR of minisatellite variable regions, showed day 14 post-transplant haemopoietic recovery to be 90% autologous in both patients. On day 30 the bone marrow showed only 1/20 and 2/18 donor metaphases. By day 100 post transplant both had 100% donor myeloid and lymphoid lineages as assessed by karyotype and minisatellite chimaerism analysis. They subsequently became RT-PCR negative for BCR-ABL. Both survive 7 and 14 months post transplant in molecular remission of CML. In one, donor T cells, stimulated with pre-transplant CML cells, induced 30-50% inhibition of pre-transplant leukaemic CFU-GM, but did not inhibit CFU-GM in the day 60 marrow (46% Ph-negative recipient, 54% donor). These results show that a non-myeloablative allotransplant can induce molecular remissions of CML through a graft-versus-leukaemia effect.

Engraftment kinetics after nonmyeloablative allogeneic peripheral blood stem cell transplantation: full donor T-cell chimerism precedes alloimmune responses.

Nonmyeloablative allogeneic stem cell transplantation has recently been explored as a safer alternative to conventional high-dose transplant regimens. Although a high incidence of mixed chimerism after nonmyeloablative procedures has been reported, the exact kinetics of engrafting donor cells in specific cellular lineages has yet to be defined. We investigated lineage-specific chimerism in 15 patients receiving an allogeneic peripheral blood stem cell (PBSC) transplant from an HLA-identical (n = 14) or a 5/6 antigen-matched sibling donor after a preparative regimen of cyclophosphamide and fludarabine. Donor chimerism was assessed weekly in T lymphocytes and myeloid cells by polymerase chain reaction (PCR) of minisatellite regions. Eight patients survived between 121 to 409 days after transplant. Ten of 14 patients surviving more than 30 days (71.4%) had delayed disease regression consistent with a graft-versus-malignancy (GVM) effect. One patient rejected the transplant with subsequent recovery of autologous hematopoiesis. Hematological recovery was rapid (median, 11 days to >/=500 neutrophils/microL) and was initially predominantly recipient in origin. Donor myeloid chimerism gradually supplanted recipient hematopoiesis and became fully donor in all survivors by 200 days after transplantation. In contrast, T-cell engraftment was more rapid, with full chimerism in 7 patients by day 30 and in 6 further patients by day 200 after cyclosporine withdrawal and donor lymphocyte infusion. Full donor T-cell engraftment preceded donor myeloid engraftment, acute graft-versus-host disease, and disease regression, consistent with a requirement for 100% donor T-cell chimerism for full expression of the alloresponse. These results emphasize the importance of lineage-specific chimerism analysis to successfully manipulate engraftment after nonmyeloablative allogeneic PBSC transplantation.

Human G-CSF-mobilized CD34-positive peripheral blood progenitor cells can stimulate allogeneic T-cell responses: implications for graft rejection in mismatched transplantation.

To investigate mechanisms of stem cell graft rejection we studied the allo-stimulatory potential of G-CSF mobilized peripheral blood progenitor cells (PBPC). CD34+ cells were purified (>95%) in a two-step procedure using immunoaffinity columns for CD34 selection and T-depletion. The capacity of CD34+ cells to stimulate allogeneic T-cell responses was compared with other cells from the same individual. CD34+ cells induced potent proliferative responses at stimulator:responder ratios of 1:20, but were approximately 50-fold less efficient compared to dendritic cells. Furthermore, CD34+ cells primed responses from partially matched allogeneic T cells in bulk cultures. Dual-colour flow cytometry showed that the co-stimulatory molecules B7.1, CD40 and ICAM-1 were absent on resting CD34-positive progenitor cells, but were induced during incubation with allogeneic lymphocytes due to a cytokine-mediated effect. Up-regulation of accessory molecules on CD34+ cells was reproduced by incubation with interferon-gamma or GM-CSF which enhanced the allo-stimulatory activity of CD34+ cells. Blocking studies with inhibitory antibodies suggested co-stimulatory functions for B7.2, ICAM-3, CD40 and LFA-3. CD34+ cells were more efficient in inducing allogeneic T-cell responses when compared to the unprocessed leukapheresis products. The reduced allo-stimulatory ability of G-CSF mobilized PBPC could be explained by the presence of CD3+ 4+ and CD3+ 8+ lymphocytes with suppressor activity. We conclude that current methods of stem cell selection for transplantation do not avoid allosensitization of the recipient and that further graft manipulation with add-back of lymphocytes or selection of subsets of CD34+ cells with reduced allo-stimulatory ability may reduce graft rejection.

Successful treatment of metastatic renal cell carcinoma with a nonmyeloablative allogeneic peripheral-blood progenitor-cell transplant: evidence for a graft-versus-tumor effect.

PURPOSE: A 50-year-old man developed progressive pulmonary metastasis resistant to interferon alfa-2b treatment 7 months after he underwent left nephrectomy for stage III renal cell carcinoma. We performed a nonmyeloablative allogeneic peripheral-blood stem-cell transplant in this patient to exploit a possible graft-versus-tumor effect from allogeneic lymphocytes. MATERIALS AND METHODS: The conditioning regimen consisted of fludarabine and cyclophosphamide followed by a T-cell replete, granulocyte-colony stimulating-factor-mobilized peripheral-blood stem-cell transplant from his HLA-identical brother. Cyclosporine was administered from days -4 to +45 to prevent graft rejection and acute graft-versus-host disease (GVHD). RESULTS: Serial polymerase chain reaction analysis of hematopoietic lineage-specific minisatellites initiallyshowed mixed chimerism in CD14(+) and CD15(+) myeloid cells, CD3(+) T cells, and CD34(+) progenitor cells, with rapid conversion to 100% donor T-cell chimerism by day +60 and 100% donor myeloid cells by day +100. Serial computed tomography scans of the chest showed stable disease at day +30, slight regression of pulmonary lesions at day +63, and complete disappearance of all pulmonary metastatic disease by day +110. Mild transient acute GVHD disease of the skin occurred on day +60 and limited chronic GVHD of the skin occurred by day +200. CONCLUSION: The complete regression of metastatic disease, which has now been maintained for more than 1 year, is compatible with a graft-versus-tumor effect.

Donor-recipient polymorphism of the proteinase 3 gene: a potential target for T-cell alloresponses to myeloid leukemia.

The curative effect of allogeneic bone marrow transplantation (BMT) is in part due to an alloresponse of donor lymphocytes against recipient leukemia termed the graft versus leukemia (GvL) effect. To identify target antigens for the GvL response on leukemia cells, we looked for polymorphism of proteinase 3, a primary granule protein overexpressed in myeloid leukemias. The study was carried out in 10 patients with hematologic diseases and their HLA-identical marrow donors. By polymerase chain reaction (PCR)-single strand conformation polymorphism assay, followed by direct sequencing of the PCR products, we found seven DNA polymorphisms. One of them encodes for either an isoleucine or a valine at position 119 of the amino acid sequence. Peptides that span the polymorphic site, at amino acids 115-124, were shown to bind in vitro to the HLA-A2 molecule. We screened 23 HLA-A2 patients with myeloid leukemia and their HLA-identical donors for this polymorphism. No relapse was found in the group of 4 evaluable patients who possessed at least one allele absent in their donor, whereas 7 of the 15 remaining evaluable patients relapsed. These data support the possibility that T-cell responses to allelic differences of proteinase 3 could be used as a basis for designing leukemia-specific adoptive T-cell therapy in acute and chronic myeloid leukemias.

Polymorphism in CD33 and CD34 genes: a source of minor histocompatibility antigens on haemopoietic progenitor cells?

Following bone marrow stem cell transplantation allo-responses against haemopoietic progenitor cells (HPC), causing graft rejection and graft-versus-leukaemia effects, can be induced by donor T cells recognizing peptides derived from polymorphic endogenous proteins present in HPC. Since CD33 and CD34 are both expressed on HPC, we looked for genetic polymorphisms that might be the source of minor histocompatibility antigens (mHA) on such cells. Bone marrow from 14 donors and their HLA-identical recipients undergoing BMT for haematological malignancies were studied. Using non-radioactive single-strand conformation polymorphism analysis (cold SSCP) of complementary DNA encoding CD33 and CD34, three DNA polymorphisms, two in CD33 and one in CD34 were found and sequenced. Two were in non-coding regions, but in CD33, ATA or ATG at codon 183 resulted in an Ile or Met in the protein sequence. Nonapeptides derived from both alleles were predicted to bind to HLA A68.1. Thus two alleles of CD33 protein exist that could be mHA. With an alternate allele frequency of < 10%, allo-responses against CD33 would be uncommon after marrow transplantation. However, donors homozygous for this allele could be used to generate cytotoxic T cells against the frequent CD33 allele, for adoptive therapy of leukaemia.

T cell-depleted bone marrow transplantation and delayed T cell add-back to control acute GVHD and conserve a graft-versus-leukemia effect.

Thirty-eight patients with hematological malignancies, received T cell-depleted marrow transplants (BMT) and cyclosporine to prevent acute graft-versus-host disease (aGVHD), followed by delayed add-back of donor lymphocytes to prevent leukemia relapse. In 26 patients scheduled for donor T cell add-back of 2 x 10(6) cells/kg on day 30 and 5 x 10(7) cells/kg on day 45 (schedule 1), the overall probability of grade > or = II aGVHD developing was 31.5%, with a 15.5% probability of aGVHD occurring after T cell add-back. In 12 patients receiving 10(7) donor T cells/kg on day 30 (schedule 2), the probability of grade > or = II aGVHD was 100%. The incidence of grade III-IV aGVHD was higher in schedule 2 than in schedule 1 (P=0.02). Of 24 evaluable patients, 10 (46%) developed chronic GVHD which was limited in eight and extensive in two. Current disease-free survival for 18 patients at standard risk for relapse (chronic myeloid leukemia (CML) in chronic or accelerated phase, acute myeloid leukemia in remission) vs 20 patients with more advanced leukemia or multiple myeloma were respectively 72% vs 12% (P < 0.01) with a 29% vs 69% probability of relapse (P=0.08). In 12 CML patients surviving more than 3 months, PCR analysis of the BCR/ABL transcript showed that minimal residual disease after T cell add-back was transient except in two patients who developed hematological relapse. Results indicate that the risk of acute GVHD is low following substantial T cell doses, transfused 45 days after transplant, using cyclosporine prophylaxis. Furthermore a graft-versus-leukemia effect was conserved.

Cytotoxic T lymphocytes specific for a nonpolymorphic proteinase 3 peptide preferentially inhibit chronic myeloid leukemia colony-forming units.

We previously showed that a peptide (PR1) derived from the primary granule enzyme proteinase 3 induced peptide specific cytotoxic T lymphocytes (CTL) in a normal HLA-A2.1+ individual. These CTL showed HLA-restricted cytotoxicity to myeloid leukemias (which overexpress proteinase 3). To further investigate their antileukemic potential, we studied the ability of PR1-specific CTL, derived from two HLA-A2.1+ normal individuals, to inhibit colony-forming unit granulocyte-macrophage (CFU-GM) from normal and leukemic individuals. CTL from 20 day PR1 peptide-pulsed lymphocyte cultures showed 89% to 98% HLA-A2.1-restricted colony inhibition of chronic myeloid leukemia targets. Colony formation in normal HLA-A2.1+ bone marrow or HLA-A2.1- CML cells was not inhibited. Sequencing of the exon encoding PR1 showed that colony inhibition was not caused by polymorphic differences in proteinase 3 between effectors and targets. Analysis by flow cytometry showed that proteinase 3 was overexpressed in the leukemia targets compared with normal marrow targets (median channel fluorescence 1,399 v 298, P = .009). These results show that PR1-specific allogeneic T cells preferentially inhibit leukemic CFU-GM based on overexpression of proteinase 3, and that proteinase 3-specific CTL could be used for leukemia-specific adoptive immunotherapy.

Radiation-enhanced expression of interferon-inducible genes in the KG1a primitive hematopoietic cell line.

X-ray treatment induces a complex molecular response in hematopoietic cells leading to cell death. Using the mRNA differential display technique, we searched for genes whose expression was modified by ionizing radiation (IR) in the human p53-deficient leukemic cell line KG1a. We isolated a partial cDNA corresponding to the interferon (IFN)-inducible 1-8d gene. The expression of both 1-8d and 9-27, another gene from the same IFN-inducible family, was increased 24 and 48 h following irradiation. We did not find enhancement of either IFNgamma mRNA or interferon regulatory factor-1 (IRF-1) mRNA in irradiated KG1a cells, indicating that 1-8d and 9-27 enhancement was not due to an IFN activation. Thus, the induction of IFN-inducible genes by IR may provide a link between radiation-induced and IFN-mediated cell death.

Ionizing radiation effects on the KG1a primitive hematopoietic cell line.

PURPOSE: Better understanding of radiation-induced effects on the hematopoietic system is important in both the context of therapeutic intervention and accidental exposure. However, direct study of these effects on the hematopoietic stem cell pool is hampered by the small number of accessible cells. We, thus, studied radiation-induced effects on the KG1a stem cell line. METHODS AND MATERIALS: We confirmed and extended the immunophenotype of KG1a with monoclonal antibodies, established a radiation survival curve, and quantified mRNAs by Northern blotting 30 min after 1, 2, and 3 Gy of ionizing radiation (IR) and followed for up to 48 h after a 3 Gy dose. Cell cycle status and apoptosis were assessed by fluorescent-activated cell sorter (FACS) analysis, cell morphology, and DNA fragmentation. RESULTS: KG1a was found to be CD34+, CD7+, Thy1 low, CD38 low, lineage negative (neg), C-KITneg and HLA-DRneg, a phenotype consistent with a primitive hematopoietic origin. This immunophenotype was not altered by x-ray irradiation. The D0 value was 1.75 Gy. We showed a time-dependent variation of c-jun mRNA expression with an early and transient dose-dependent induction followed by a second increase at 24 and 48 h: a biphasic dose-dependent variation of bcl-2 expression 30 min after irradiation with a reduction of mRNA level at 1 Gy, and a normalization at higher doses and stable levels of mRNA for c-fos, c-myc, G-CSF, GM-CSF, IL-6, TNF-alpha, TGF-beta, and MIP-1 alpha genes. Cell cycle analysis showed the absence of G1/S phase arrest, a point consistent with the absence of detection of P53 mRNA by Northern blot analysis. The dose-dependent G2/M phase arrest was not followed by significant apoptotic cell death. CONCLUSION: Taken together, this data indicates that radiation-induced cell death of KG1a, a cell line that has a relatively high D0 value, does not seem to be the result of the apoptotic pathway but occurs subsequent to a G2/M phase arrest.