Human adenoviral (HAdV) chronic arthritis expands the infectious spectrum of primary agammaglobulinemia.

Inborn errors of immunity (IEI) are a heterogeneous entity with an increasing number of late diagnoses. Besides infections, inflammatory manifestations are a growing part of the clinical landscape of IEI. These complications are of unknown causes and often lead to the prescription of immunosuppressive agents that worsen the underlying immune defect. We here report the case of an adult patient diagnosed with chronic Human Adenovirus C-1 arthritis in the setting of primary agammaglobulinemia. Metagenomic next-generation sequencing led to the correct diagnosis and high-dose intravenous immunoglobulins resulted in complete recovery. This observation gives new insights into adenoviral immunity and underlines the importance of metagenomics in the diagnosis of inflammatory manifestations in immunocompromised patients.

Impact of acute and chronic graft-versus-host disease on human B-cell generation and replication.

Using B-cell rearrangement excision circle measurements, we analyzed B-cell reconstitution in a cohort of 243 patients who underwent allogeneic stem cell transplantation. Acute and chronic graft-versus-host disease (aGVHD and cGVHD, respectively) transiently increased B-cell replication but decreased overall B-cell neogenesis with a clear difference in terms of kinetics. Moreover, the impact of aGVHD in the absence of cGVHD was transient, recovering at month 6 similar values as in patients who did not suffer from GVHD. Conversely, impact of cGVHD at month 12 in multivariate analysis was independent of the previous aGVHD effect on B-cell output. Finally, we showed in patients affected with cGVHD a higher B-cell division rate that correlates with an elevated BAFF/CD19(+) B-cell ratio, supporting a B-cell hyperactivation state in vivo.

Binding of HLA-G to ITIM-bearing Ig-like transcript 2 receptor suppresses B cell responses.

Inhibition of B cells constitutes a rational approach for treating B cell-mediated disorders. We demonstrate in this article that the engagement of the surface Ig-like transcript 2 (ILT2) inhibitory receptor with its preferential ligand HLA-G is critical to inhibit B cell functions. Indeed, ILT2-HLA-G interaction impedes both naive and memory B cell functions in vitro and in vivo. Particularly, HLA-G inhibits B cell proliferation, differentiation, and Ig secretion in both T cell-dependent and -independent models of B cell activation. HLA-G mediates phenotypic and functional downregulation of CXCR4 and CXCR5 chemokine receptors on germinal center B cells. In-depth analysis of the molecular mechanisms mediated by ILT2-HLA-G interaction showed a G0/G1 cell cycle arrest through dephosphorylation of AKT, GSK-3ß, c-Raf, and Foxo proteins. Crucially, we provide in vivo evidence that HLA-G acts as a negative B cell regulator in modulating B cell Ab secretion in a xenograft mouse model. This B cell regulatory mechanism involving ILT2-HLA-G interaction brings important insight to design future B cell-targeted therapies aimed at reducing inappropriate immune reaction in allotransplantation and autoimmune diseases.

Long-term immune reconstitution and infection burden after mismatched hematopoietic stem cell transplantation.

Mismatched unrelated donor (MMUD) or umbilical cord blood (UCB) can be chosen as alternative donors for allogeneic stem cell transplantation but might be associated with long-lasting immune deficiency. Sixty-six patients who underwent a first transplantation from either UCB (n = 30) or 9/10 MMUD (n = 36) and who survived beyond 3 months were evaluated. Immune reconstitution was prospectively assessed at sequential time points after transplantation. NK, B, CD4(+), and CD8(+) T cells and their naïve and memory subsets, as well as regulatory T cells (Treg), were studied. Detailed analyses on infections occurring after 3 months were also assessed. The 18-month cumulative incidences of infection-related death were 8% and 3%, and of infections were 72% and 57% after MMUD and UCB transplantation, respectively. Rates of infection per 12 patient-month were roughly 2 overall (1 for bacterial, .9 for viral, and .3 for fungal infections). Memory, naïve CD4(+) and CD8(+)T cells, naïve B cells, and Treg cells reconstitution between the 2 sources were roughly similar. Absolute CD4(+)T cells hardly reached 500 per µL by 1 year after transplantation and most B cells were of naïve phenotype. Correlations between immune reconstitution and infection were then performed by multivariate analyses. Low CD4(+) and high CD8(+)T cells absolute counts at 3 months were linked to increased risks of overall and viral (but not bacterial) infections. When assessing for the naïve/memory phenotypes at 3 months among the CD4(+) T cell compartment, higher percentages of memory subsets were protective against late infections. Central memory CD4(+)T cells protected against overall and bacterial infections; late effector memory CD4(+)T cells protected against overall, bacterial, and viral infections. To the contrary, high percentage of effector- and late effector-memory subsets at 3 months among the CD8(+) T cell compartment predicted higher risks for viral infections. Patients who underwent transplantation from alternative donors represent a population with very high risk of infection. Detailed phenotypic analysis of immune reconstitution may help to evaluate infection risk and to adjust infection prophylaxis.

Favorable impact of natural killer cell reconstitution on chronic graft-versus-host disease and cytomegalovirus reactivation after allogeneic hematopoietic stem cell transplantation.

Natural killer cells are the first lymphocyte subset to reconstitute, and play a major role in early immunity after allogeneic hematopoietic stem cell transplantation. Cells expressing the activating receptor NKG2C seem crucial in the resolution of cytomegalovirus episodes, even in the absence of T cells. We prospectively investigated natural killer-cell reconstitution in a cohort of 439 adult recipients who underwent non-T-cell-depleted allogeneic hematopoietic stem cell transplantation between 2005 and 2012. Freshly collected blood samples were analyzed 3, 6, 12 and 24 months after transplantation. Data were studied with respect to conditioning regimen, source of stem cells, underlying disease, occurrence of graft-versus-host disease, and profiles of cytomegalovirus reactivation. In multivariate analysis we found that the absolute numbers of CD56(bright) natural killer cells at month 3 were significantly higher after myeloablative conditioning than after reduced intensity conditioning. Acute graft-versus-host disease impaired reconstitution of total and CD56(dim) natural killer cells at month 3. In contrast, high natural killer cell count at month 3 was associated with a lower incidence of chronic graft-versus-host disease, independently of a previous episode of acute graft-versus-host disease and stem cell source. NKG2C(+)CD56(dim) and total natural killer cell counts at month 3 were lower in patients with reactivation of cytomegalovirus between month 0 and month 3, but expanded greatly afterwards. These cells were also less numerous in patients who experienced later cytomegalovirus reactivation between month 3 and month 6. Our results advocate a direct role of NKG2C-expressing natural killer cells in the early control of cytomegalovirus reactivation after allogeneic hematopoietic stem cell transplantation.

Severe Altered Immune Status After Burn Injury Is Associated With Bacterial Infection and Septic Shock.

Introduction: Burn injury is associated with a high risk of death. Whether a pattern of immune and inflammatory responses after burn is associated with outcome is unknown. The aim of this study was to explore the association between systemic immune and inflammatory responses and outcome in severely-ill burn patients. Materials and Methods: Innate immunity, adaptive immunity, activation and stress and inflammation biomarkers were collected at admission and days 2, 7, 14, and 28 in severely-ill adult burn patients. Primary endpoint was mortality at day 90, secondary endpoint was secondary infections. Healthy donors (HD) served as controls. Multiple Factorial Analysis (MFA) was used to identify patterns of immune response. Results: 50 patients were included. Age was 49.2 (44.2-54.2) years, total burn body surface area was 38.0% (32.7-43.3). Burn injury showed an upregulation of adaptive immunity and activation biomarkers and a down regulation of innate immunity and stress/inflammation biomarkers. High interleukin-10 (IL-10) at admission was associated with risk of death. However, no cluster of immune/inflammatory biomarkers at early timepoints was associated with mortality. HLA-DR molecules on monocytes at admission were associated with bacterial infections and septic shock. Later altered immune/inflammatory responses in patients who died may had been driven by the development of septic shock. Conclusion: Burn injury induced an early and profound upregulation of adaptive immunity and activation biomarkers and a down regulation of innate immunity and stress/inflammation biomarkers. Immune and inflammatory responses were associated with bacterial infection and septic shock. Absence of immune recovery patterns was associated with poor prognosis.

Daoy medulloblastoma cells that express CD133 are radioresistant relative to CD133- cells, and the CD133+ sector is enlarged by hypoxia.

PURPOSE: Primary medulloblastoma and glioblastoma multiforme tumor cells that express the surface marker CD133 are believed to be enriched for brain tumor stem cells because of their unique ability to initiate or reconstitute tumors in immunodeficient mice. This study sought to characterize the radiobiological properties and marker expression changes of CD133+ vs. CD133- cells of an established medulloblastoma cell line. METHODS AND MATERIALS: Daoy and D283 Med cell lines were stained with fluorescently labeled anti-CD133 antibody and sorted into CD133+ and CD133- populations. The effect of oxygen (2% vs. 20%) on CD133 expression was measured. Both populations were analyzed for marker stability, cell cycle distribution, and radiosensitivity. RESULTS: CD133+ Daoy cells restored nearly native CD133+ and CD133- populations within 18 days, whereas CD133- cells remained overwhelmingly CD133-. Culturing Daoy cells in 2% oxygen rather than the standard 20% oxygen increased their CD133 expression 1.6-fold. CD133+ Daoy cells were radioresistant via the beta-parameter of the linear-quadratic model relative to CD133- Daoy cells, although their alpha-parameters and cell cycle distributions were identical. CONCLUSIONS: Restoration of the original CD133+ and CD133- populations from CD133+ Daoy cells in serum is further evidence that CD133+ cells are functionally distinct from CD133- cells. The radioresistance of CD133+ compared with CD133- Daoy cells is consistent with better repair of sublethal damage. Enlargement of the CD133+ sector is a new feature of the hypoxic response.

Long-term immune reconstitution and T cell repertoire analysis after autologous hematopoietic stem cell transplantation in systemic sclerosis patients.

The determinants of clinical responses after autologous hematopoietic stem cell transplantation (aHSCT) in systemic sclerosis (SSc) are still unraveled. We analyzed long-term immune reconstitution (IR) and T cell receptor (TCR) repertoire diversity in 10 SSc patients, with at least 6 years simultaneous clinical and immunological follow-up after aHSCT. Patients were retrospectively classified as long-term responders (A, n = 5) or non-responders (B, n = 5), using modified Rodnan's skin score (mRSS) and forced vital capacity (FVC%). All patients had similar severe SSc before aHSCT. Number of reinjected CD34+ cells was higher in group B versus A (P = 0.02). Long-term mRSS fall >25% was more pronounced in group A (P = 0.004), the only to improve long-term FVC% >10% (P = 0.026). There was an overall trend toward increased of T cell reconstitution in group B versus A. B cells had a positive linear regression slope in group A (LRS = 11.1) and negative in group B (LRS = -11.6). TCR repertoire was disturbed before aHSCT and the percentage of polyclonal families significantly increased at long-term (P = 0.046), with no difference between groups. Despite improved skin score after aHSCT in all SSc patients, pretransplant B cell clonal expansion and faster post-transplant T cell IR in long-term non-responder/relapsing patients call for new therapeutic protocols guided by IR analysis to improve their outcome.

Clinicopathological significance of major histocompatibility complex class I-related chain a and B expression in thyroid cancer.

CONTEXT: Major histocompatibility complex class I-related chains A and B (MICA/B) are two stress-inducible ligands for the immunoreceptor NKG2D that is expressed on cytotoxic T cells and natural killer (NK) cells. It is not known whether MICA/B expression is up-regulated in thyroid cancer as a result of oncogene activation. OBJECTIVE: The objective of the investigation was to study MICA/B expression and regulation in thyroid cancer and its role in mediating the cytotoxicity of NK cells. METHODS: MICA/B expression in thyroid cancer was analyzed by immunohistochemical staining. Cell surface MICA/B levels in thyroid tumor cell lines and fresh tumor cells were analyzed by flow cytometric analysis. The susceptibility of thyroid tumor cells to NK cell killing was tested by using (51)Cr release assay. RESULTS: MICA/B was expressed at moderate or high levels in 18 of 39 papillary thyroid carcinomas and four of eight anaplastic thyroid carcinomas. MICA/B expression was confirmed by flow cytometric analysis in three fresh thyroid neoplasms. MICA/B expression was detected in eight of 10 thyroid tumor cell lines and correlated with their sensitivity to killing by the NKG2/D-positive NK-92 cells. Blocking of NKG2D and MICA/B interaction by specific antibodies partially led to the inhibition of NK-92 cell-mediated cytotoxicity. The MAPK inhibitors were able to block MICA/B expression in MRO87 and HeLa cells. Transient transfection of mutant BRAF and RAS oncogenes led to increased MICA/B expression in 293 cells and WRO82 cells. CONCLUSION: MICA/B expression is up-regulated in thyroid cancer, probably due to the activation of the MAPK pathway. MICA/B in thyroid cancer plays an important role in NK-92 cell-mediated cytotoxicity.

Ipilimumab reshapes T cell memory subsets in melanoma patients with clinical response.

PURPOSE: Therapy targeting CTLA-4 immune checkpoint provides increased survival in patients with advanced melanoma. However, immunotherapy is frequently associated with delayed and heterogeneous clinical responses and it is important to identify prognostic immunological correlates of clinical endpoints. EXPERIMENTAL DESIGN: 77 patients with stage III/IV melanoma were treated with ipilimumab alone every 3 weeks, during 9 weeks. Blood samples were collected at the baseline and before each dose for in depth immune monitoring. RESULTS: The median follow-up was 28 mo with a median survival of 7 mo. Survival and clinical benefit were significantly improved when absolute lymphocyte count at the baseline was above 1 × 10(9)/L. Notably, ipilimumab had a global effect on memory T cells, with an early increase of central and effector subsets in patients with disease control. By contrast, percentages of stem cell memory T cells (TSCM) gradually decreased despite stable absolute counts and sustained proliferation, suggesting a process of differentiation. Higher proportions of eomes(+) and Ki-67(+) T cells were observed, with enhanced skin homing potential and induction of cytotoxic markers. CONCLUSION: These results suggest that CTLA-4 blockade is able to reshape the memory subset with the potential involvement of Eomes and memory subsets including TSCM.

CD158k is a reliable marker for diagnosis of Sézary syndrome and reveals an unprecedented heterogeneity of circulating malignant cells.

The diverse aspects of cutaneous T-cell lymphomas may impede the diagnosis of Sézary syndrome (SS) and mycosis fungoides (MF), in particular, at early stages of the disease. We defined the CD158k/KIR3DL2 molecule as a first positive cell surface marker for Sézary cells (SCs). Here, we designed an optimized flow cytometry gating strategy, allowing the definition of lymphocytes of different sizes and defects of cell surface markers. Quantification by cytomorphology, flow cytometry, or clonal evaluation, gave similar results at initial time points and during the evolution in a prospective study involving 64 consecutive cutaneous T-cell lymphoma or erythrodermic patients. We found that CD158k+ T cells and circulating CD4+ T cells from MF patients exhibited unexpected patterns of cell surface expression with a marked heterogeneity of circulating lymphocytes even at initial diagnosis. Taken together, our results show that a multistep gating of CD158k+ cells is reliable to assess tumor burden in case of SS and suggest that both circulating MF CD4+ T cells and CD158k+ T cells are not homogeneous distinct memory populations. Further phenotypic and functional characterizations of such subsets are needed to better understand the underlying molecular mechanisms leading to the development of these diseases.

Expression of a CD20-specific chimeric antigen receptor enhances cytotoxic activity of NK cells and overcomes NK-resistance of lymphoma and leukemia cells.

Despite the clinical success of CD20-specific antibody rituximab, malignancies of B-cell origin continue to present a major clinical challenge, in part due to an inability of the antibody to activate antibody-dependent cell-mediated cytotoxicity (ADCC) in some patients, and development of resistance in others. Expression of chimeric antigen receptors in effector cells operative in ADCC might allow to bypass insufficient activation via FcgammaRIII and other resistance mechanisms that limit natural killer (NK)-cell activity. Here we have generated genetically modified NK cells carrying a chimeric antigen receptor that consists of a CD20-specific scFv antibody fragment, via a flexible hinge region connected to the CD3zeta chain as a signaling moiety. As effector cells we employed continuously growing, clinically applicable human NK-92 cells. While activity of the retargeted NK-92 against CD20-negative targets remained unchanged, the gene modified NK cells displayed markedly enhanced cytotoxicity toward NK-sensitive CD20 expressing cells. Importantly, in contrast to parental NK-92, CD20-specific NK cells efficiently lysed CD20 expressing but otherwise NK-resistant established and primary lymphoma and leukemia cells, demonstrating that this strategy can overcome NK-cell resistance and might be suitable for the development of effective cell-based therapeutics for the treatment of B-cell malignancies.