Production and persistence of specific antibodies in COVID-19 patients with hematologic malignancies: role of rituximab.

The ability of patients with hematologic malignancies (HM) to develop an effective humoral immune response after COVID-19 is unknown. A prospective study was performed to monitor the immune response to SARS-CoV-2 of patients with follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), chronic lymphoproliferative disorders (CLD), multiple myeloma (MM), or myelodysplastic/myeloproliferative syndromes (MDS/MPN). Antibody (Ab) levels to the SARS-CoV-2 nucleocapsid (N) and spike (S) protein were measured at +1, +3, +6 months after nasal swabs became PCR-negative. Forty-five patients (9 FL, 8 DLBCL, 8 CLD, 10 MM, 10 MDS/MPS) and 18 controls were studied. Mean anti-N and anti-S-Ab levels were similar between HM patients and controls, and shared the same behavior, with anti-N Ab levels declining at +6 months and anti-S-Ab remaining stable. Seroconversion rates were lower in HM patients than in controls. In lymphoma patients mean Ab levels and seroconversion rates were lower than in other HM patients, primarily because all nine patients who had received rituximab within 6 months before COVID-19 failed to produce anti-N and anti-S-Ab. Only one patient requiring hematological treatment after COVID-19 lost seropositivity after 6 months. No reinfections were observed. These results may inform vaccination policies and clinical management of HM patients.

Two siblings presenting with novel ADA2 variants, lymphoproliferation, persistence of large granular lymphocytes, and T-cell perturbations.

The presence of large granular lymphocytes has been reported in patients with ADA2 deficiency and T-LGL leukemia. Here we describe two siblings with novel ADA2 variants, expanding the mutational spectrum of ADA2 deficiency. We show that lymphoproliferation, persistence of large granular lymphocytes, T-cell perturbations, and activation of PI3K pathway, measured by means of phosphorylation levels of S6, are detectable in DADA2 patients without T-LGL leukemia.

Lymphocyte homeostasis is maintained in perinatally HIV-infected patients after three decades of life.

BACKGROUND: While immunosenescence, defined as reduced production of new lymphocytes, restriction of T-cell receptor repertoire and telomeres shortening, has been extensively evaluated in HIV-infected children and adults, no data about these parameters are available in perinatally-infected patients with very long-lasting HIV infection. METHODS: We compared thymic and bone marrow output, telomere length (measured by Real-Time PCR) and T-cell receptor repertoire (determined by spectratyping) of 21 perinatally HIV-infected subjects (with a median of 27 years of infection) with those of 19 age-matched non-perinatally HIV-infected patients and 40 healthy controls. All patients received a combined antiretroviral therapy. RESULTS: While thymic and bone marrow output were not different among the analyzed groups, telomere length in peripheral blood cells and T-cell receptor diversity were significantly lower in HIV-perinatally and non-perinatally infected individuals compared to healthy controls. CONCLUSIONS: In HIV-infected subjects, a normal thymic output together with a reduced telomere length and a restricted T-cell receptor repertoire could be explained by the shift of newly produced cells into memory subsets. This phenomenon may allow to control viral infection and maintain peripheral homeostasis.

Peripheral loss of regulatory T cells and polyautoimmunity in an HIV-infected patient.

We report a case of a human immunodeficiency virus (HIV)/hepatitis C virus-co-infected patient with an optimal virological status but with a poor CD4+ cell profile, followed up in the University Department of Infectious and Tropical Diseases of Brescia, Italy. He presented several autoimmune diseases (ADs) over the years concomitant with CD4+ cell increase episodes following severe immune depression of unknown cause. We studied T- and B-cell subsets and found low levels of K-deleting Recombination Excision Circles, T-cell Receptor Excision Circles and B and T memory subpopulations, which indicated that the bone marrow and thymic outputs were lower than in healthy controls. The most relevant phenotypic alteration was in the regulatory T-cell (Treg) population, because total Tregs as well as naïve, central memory and effector memory cells were detected at very low levels. This was the first case of polyautoimmunity defined as the presence of more than one AD in the same individual, occurring in an HIV patient. Several factors may be implicated, including genetic susceptibility, environmental factors, concomitant therapies and dysregulation of immune system cells. The extremely low number of Tregs found in our patient may play a major role in the regulation of the immune response and the development of all ADs.

Newly produced T and B lymphocytes and T-cell receptor repertoire diversity are reduced in peripheral blood of fingolimod-treated multiple sclerosis patients.

BACKGROUND: Fingolimod inhibits lymphocyte egress from lymphoid tissues, thus altering the composition of the peripheral lymphocyte pool of multiple sclerosis patients. OBJECTIVE: The objective of this paper is to evaluate whether fingolimod determines a decrease of newly produced T- and B-lymphocytes in the blood and a reduction in the T-cell receptor repertoire diversity that may affect immune surveillance. METHODS: Blood samples were obtained from multiple sclerosis patients before fingolimod therapy initiation and then after six and 12 months. Newly produced T and B lymphocytes were measured by quantifying T-cell receptor excision circles and K-deleting recombination excision circles by real-time PCR, while recent thymic emigrants, naive CD8(+) lymphocytes, immature and naive B cells were determined by immune phenotyping. T-cell receptor repertoire was analyzed by complementarity determining region 3 spectratyping. RESULTS: Newly produced T and B lymphocytes were significantly reduced in peripheral blood of fingolimod-treated patients. The decrease was particularly evident in the T-cell compartment. T-cell repertoire restrictions, already present before therapy, significantly increased after 12 months of treatment. CONCLUSIONS: These results do not have direct clinical implications but they may be useful for further understanding the mode of action of this immunotherapy for multiple sclerosis patients.

Reduction of peripheral blood T cells producing IFN-γ and IL-17 after therapy with abatacept for rheumatoid arthritis.

OBJECTIVES: Abatacept (ABA), a molecule used in the treatment of rheumatoid arthritis (RA), competes with the engagement of CD28, a T-cell receptor for co-stimulatory signals. CD28-mediated signalling regulates several T-cell functions, including inflammatory cytokine production and regulatory T cells (Treg) differentiation. Therefore, our objective was to evaluate the effects of ABA on peripheral blood T-lymphocyte cytokine production and on the number of circulating Treg. METHODS: In 24 RA patients treated with ABA for at least 6 months the proportions and absolute numbers of peripheral blood T cells producing interferon-gamma (IFN-γ) and interleukin-17 (IL-17) after in vitro stimulation, as well as those of Treg were longitudinally evaluated by flow cytometry. RESULTS: At baseline, compared with 16 healthy controls, RA patients had a higher percentage of CD4+ and CD8+ T cells producing IL-17 (p=0.021, and p=0.006, respectively), as well as of circulating Treg (p=0.041). After 6 months of therapy with ABA, there was a decrease of the percentage of IFN-γ- and IL-17-producing CD8+ T cells (p=0.033 and p=0.035, respectively), and of Treg (p=0.008), while that of IL-17-producing CD4+ T cells decreased after 12 months of treatment (p=0.005). The number of IL-17-producing T cells and of Treg, higher than in controls at baseline, normalised after ABA therapy. All these variations were statistically significant only in RA patients with EULAR good clinical response (n=17). CONCLUSIONS: The blockade of CD28 signal caused by ABA induces the decrease in peripheral blood of IL-17- and IFN-γ-producing T cells.

Modulation of the central memory and Tr1-like regulatory T cells in multiple sclerosis patients responsive to interferon-beta therapy.

BACKGROUND: Interferon-beta is used to reduce disease activity in multiple sclerosis, but its action is incompletely understood, individual treatment response varies among patients, and biological markers predicting clinical benefits have yet to be identified. Since it is known that multiple sclerosis patients have a deficit of the regulatory T-cell subsets, we investigated whether interferon-beta therapy induced modifications of the two main categories of regulatory T cells (Tregs), natural and IL-10-secreting inducible Tr1 subset, in patients who are biologically responsive to the therapy. METHODS: T-cell phenotype was determined by flow cytometry, while real-time PCR was used to evaluate interferon-beta bioactivity through MxA determination, and to measure the RNA for IL-10 and CD46 molecule in peripheral blood mononuclear cells stimulated with anti-CD46 and anti-CD3 monoclonal antibodies, which are known to expand a Tr1-like population. RESULTS: Interferon-beta induced a redistribution of natural Treg subsets with a shift of naive Tregs towards the 'central memory-like' Treg population that expresses the CCR7 molecule required for the in vivo suppressive activity. Furthermore, in a subgroup of treated patients, the CD46/CD3 co-stimulation, probably through the Tr1-like subset modulation, increased the production of RNA for IL-10 and CD46. The same group showed a lower median EDSS score after two years of therapy. CONCLUSIONS: The selective increase of 'central memory-like' subset and the involvement of the Tr1-like population may be two of the mechanisms by which interferon-beta achieves its beneficial effects. The quantification of RNA for IL-10 and CD46 could be used to identify patients with a different response to interferon-beta therapy.

Adult-type hypolactasia genotyping in Northern Italy: prevalence of C/T-13910 polymorphism and questions after comparison with existing data.

A genotyping assay was setup to assess the prevalence, in the population of a Northern Italian city, of the C/T-13910 single nucleotide polymorphism, closely associated to lactose malabsorption in many world areas including Sardinia. The results were compared to published Italian data, in order to evaluate the worth of a future validation of the assay for use in routine practice. DNA was extracted from blood samples of 123 randomly chosen healthy blood donors coming from the same city area, and was analyzed by a real-time polymerase chain reaction (PCR) genotyping assay; the frequency of the hypolactasia-associated CC-genotype was compared to the weighted average of results extracted from studies reporting the frequency of hypolactasic phenotype or genotype in nearby or distant Italian regions. Sixty-five percent of donors carried the CC-genotype, a percentage similar to other northern Italian cities, but significantly higher than what previously determined in surrounding Italian regions at the phenotype level, i.e. by breath test. This discrepancy parallels recent reports of non concordance between results of genotyping and hypolactasic phenotype in some world areas, including a neighbouring Northern Italian city. A north-south gradient of CC-prevalence was also observed. These results reinforce the notion of wide inter-regional variations in the frequency of C/T-13910 polymorphism and of incostant concordance with hypolactasic phenotype, even in subjects from the same country. Given the unsatisfactory results recently obtained from validation of a related assay in a neighbouring city, the authors decided not to proceed further and keep the assay only as a diagnostic aid in special situations.

Renewal of the T-cell compartment in multiple sclerosis patients treated with glatiramer acetate.

The immunomodulating activity of glatiramer acetate on T-cells of multiple sclerosis patients has only been partially clarified. The objective of this work was to investigate whether glatiramer acetate modifies thymic release of newly produced T-cells and the peripheral composition of the T-cell repertoire. T-cell receptor excision circles, (thymic) naive (CD4(+)CD45RA(+)CCR7(+)CD31(+)) T helper cells, and central (CD4(+)CD45RA(-)CCR7(+)) and effector (CD4(+)CD45RA(-)CCR7(-)) memory T-cells were evaluated in 89 untreated patients, 84 patients treated for at least 1 year, and 31 patients beginning treatment at the time of inclusion in the study and then followed-up for 12 months; controls were 81 healthy donors. The T-cell repertoire was analysed in selected samples. The percentage of (thymic)naive T helper cells was diminished in untreated patients, but rose to control values in treated subjects; a decrease in central memory T-cells was also observed in treated patients. Follow-up patients could be divided into two subgroups, one showing unmodified (thymic)naive T helper cells and T-cell diversity, the other in which the increased release of new T-cells was accompanied by modifications of the T-cell repertoire. Glatiramer acetate modifies the peripheral T-cell pool by activating a thymopoietic pathway of T-cell release that leads to a different setting of T-cell diversity and, likely, to a dilution of autoreactive T-cells.

Interferon-beta therapy monitoring in multiple sclerosis patients.

Interferon-beta (IFN-beta) therapy has a central place in the management of multiple sclerosis (MS). The three recombinant IFN-beta preparations currently available have shown benefit on activity measures (relapses and active lesions apparent on magnetic resonance imaging), while therapy advantages on progression measures (disability and total lesion burden) are less consistent. Moreover, IFN-beta is effective only in a percentage of patients, since in many of them neutralizing anti-IFN-beta antibodies develop after 6-18 months of treatment, leading to loss of drug bioactivity. Comparative data across studies made with different IFN-beta preparations suggest that the optimal choice of IFN-beta subtype, preparation and dose regimen are important determinants of efficacy. Because IFN-beta actions depend on the activation of IFN-inducible genes, in addition to the direct quantification of anti-IFN-beta antibodies, several other methods for the measure of IFN-beta biologic activity have been recently developed. Among these, the determination of the IFN-beta-inducible gene product Myxovirus protein A (MxA) has proven to be the most reliable one. Another still open point is the role of the differential expression of IFN-beta receptor (IFNAR) components, since IFNAR2 subunit can be synthesized in three isoforms: functional, truncated non-functional and soluble. While this and other important issues require further studies, this article reviews and discusses the importance, potential and limits of the methods currently available to monitor IFN-beta therapy in MS patients.

Characterization of T-cell population in children with prolonged fetal exposure to dexamethasone for anti-Ro/SS-A antibodies associated congenital heart block.

The objectives of the study were to characterize the production, function and survival of T lymphocytes of children with prolonged fetal exposure to dexamethasone for anti-Ro/SS-A antibodies associated congenital complete heart block. The analysis of thymic function, studied by measuring the level of T-cell receptor excision circles, was performed by real time PCR, the composition of T-cell subpopulation was evaluated by flow cytometry and the T-cell diversity was assayed by heteroduplex analysis. T-cell competence was gauged at two functional levels by determining the proliferation and the number of T-cell divisions and by measuring gamma-interferon production after mitogenic stimulation. We observed that the thymic output, distribution of T-cell subsets, thymidine incorporation, number of T-cell divisions, and y-interferon production were comparable to those of age-matched control. On the contrary, heteroduplex analysis demonstrated the presence of both polyclonal and oligoclonal peripheral T-cell repertoires. In conclusion, the analysis of the T-cell compartment in children with prolonged intrauterine exposure to high dose dexamethasone did not disclose any relevant abnormality, except a restriction of T-cell receptor diversity in some patients.

Stem cell transplantation for the Wiskott-Aldrich syndrome: a single-center experience confirms efficacy of matched unrelated donor transplantation.

The treatment of Wiskott-Aldrich syndrome (WAS), a once uniformly fatal disorder, has evolved considerably as the use of hematopoietic stem cell transplant has become more widespread. For the majority of patients who lack an human leukocyte antigen-identical sibling, closely matched unrelated donor bone marrow transplant (MUD BMT) at an early age is an excellent option that nevertheless is not uniformly chosen. We retrospectively analyzed our experience with transplantation in 23 patients with WAS from 1990 to 2005 at the University of Brescia, Italy, of whom 16 received MUD BMT. Myeloablative chemotherapy was well tolerated with median neutrophil engraftment at day 18, and no cases of grade III or IV graft-vs-host disease. Overall survival was very good with 78.2% (18/23) of the whole cohort and 81.2% (13/16) of MUD BMT recipients surviving. Among 18 survivors, full donor engraftment was detected in 12 patients, and stable mixed chimerism in all blood lineages in four patients. Deaths were limited to patients who had received mismatched related BMT or who had severe clinical symptomatology at the time of transplantation, further emphasizing the safety and efficacy of MUD BMT when performed early in the clinical course of WAS.

Hematopoietic stem cell transplantation in Omenn syndrome: a single-center experience.

We retrospectively analyzed the outcome of hematopoietic stem cell transplantations (HSCT) performed at our Center between 1991 and 2002 in 11 unselected patients with Omenn syndrome, a variant of severe combined immunodeficiency. The patients' mean age at the time of the first HSCT was 8.4 months. Two patients received two, and one patient three, HSCT procedures. The resulting 15 HSCT derived in seven cases from HLA-haploidentical parents, in four patients from matched unrelated donors, in three cases from an HLA phenotypically identical related donor, and in one case from an HLA genotypically identical family donor. Nine out of 11 patients are alive and immunoreconstituted 30-146 months after transplantation. At the time of the most recent evaluation, all of the nine survivors had normal T-cell function, and eight of them had developed normal antibody production. This study demonstrates an overall mortality of 18.2%, which is substantially lower than previously reported. Early recognition of OS, rapid initiation of adequate supportive treatment and HSCT lead to improved outcome for this otherwise fatal disease, regardless of the origin and matching of hematopoietic stem cells.

Isolation, purification, and heterogeneity of human lymphatic endothelial cells from different tissues.

Relatively few attempts have been made in the past to isolate and expand lymphatic endothelial cells (LECs). Recently this task has become feasible thanks to the identification of new lymphatic markers such as Podoplanin, Lyve-1, Prox-1 and D2-40. Using a two-step purification method based on the sorting of endothelial cells with Ulex Europaeus Agglutinin 1-coated beads followed by purification with monoclonal antibody D2-40, we were able to purify and in vitro expand human derived LECs from tissues such as lymph node, spleen, thymus, palatine tonsil and iliac lymphatic vessels. The isolated LECs were expanded on collagen type 1 and fibronectin coated flasks for up to 8-10 passages and then analyzed for phenotypic and functional properties. LECs were able to form a capillary like network, when seeded on Cultrex BME, indicating their capability to form lymphatic vessels in vitro. Comparative studies were performed, and we found that specific lymphatic and vascular markers were differentially expressed by LECs prepared from different sources, clearly demonstrating the phenotypic heterogeneity of LECs from different organs and different segments of the lymphatic vasculature. We here propose a new technique to make available ready sources of abundant well-characterized human LECs to examine normal profiles and behavior to compare with abnormal conditions.

Characterization of CD20-transduced T lymphocytes as an alternative suicide gene therapy approach for the treatment of graft-versus-host disease.

We have previously proposed the CD20 molecule as a novel suicide gene for T lymphocytes in the context of allogeneic bone marrow transplantation, because CD20 can be used both as a selection marker and as a killer gene after exposure to the anti-CD20 therapeutic antibody rituximab. We now report on preclinical studies using this novel system, in which the best transduction protocol, reproducibility, yield, feasibility, and functionality of the transduced T lymphocytes have been investigated with a large donor series. Wild-type human CD20 cDNA was transduced into human T lymphocytes, using a Moloney-derived retroviral vector. Alternative protocols were tested by employing either one or four spinoculations (in which cells are centrifuged in the presence of retroviral vector supernatant) and stimulating T cells with phytohemagglutinin (PHA) or anti-CD3/CD28. One spinoculation alone was sufficient to obtain approximately 30% CD20-positive cells within four experimental days. Four spinoculations significantly increased transduction to 60%. A small difference in transduction efficiency was observed between the two stimulation methods, with PHA being superior to anti-CD3/CD28. Transduced cells could be purified on immunoaffinity columns, with purity reaching 98% and yield being on average 50%. Finally, 86-97% of immunoselected T lymphocytes could be killed in vitro with rituximab and complement. More importantly, the CD20 transgene did not alter the functionality of T lymphocytes with respect to allogeneic recognition and cytotoxic response, anti-Epstein-Barr virus cytotoxic response, antigenic response to tetanus toxoid antigen, interleukin 2 (IL-2), IL-4, and interferon gamma production; chemotaxis in the presence of stromal cell-derived factor 1, phenotype for several activation markers including HLA-DR, CD25, CD69, and CD95, and T cell repertoire.

Low thymic output and reduced heterogeneity of alpha/beta, but not gamma/delta, T lymphocytes in infants with ataxia-telangiectasia.

Ataxia-telangiectasia, a genetic disease caused by the homozygous mutation of the ATM gene, is frequently associated to a deficit of humoral and cellular immune functions. A decreased thymic output and skewed T cell and B cell receptor repertoires have been recently described in children over 7 years of age and in adults with this disease and have been proposed as a possible explanation for the immunodeficiency. To understand whether T cell defects arise early in life as a consequence of ATM gene mutations, we analysed the extent of thymic function by measuring the number of naïve T cells and by studying the heterogeneity of T cells by means of heteroduplex analysis, in two children less than 2 years old with a remarkable reduction of T cell count. We found that the thymic output is decreased in babies with ataxia-telangiectasia if compared with that observed in age-matched normal babies. The low production of new T cells is associated to a reduction of the diversity of alpha/beta, but not gamma/delta, T lymphocytes. Our data indicate that ATM mutation limits the generation of a wide alpha/beta T cell repertoire and this feature can be responsible for the immunodeficiency observed in ataxia-telangiectasia babies.

Impaired thymic output and restricted T-cell repertoire in two infants with immunodeficiency and early-onset generalized dermatitis.

We evaluated the T-cell repertoire and the thymic output in two infants, one with Omenn Syndrome (OS) and another with complete DiGeorge Syndrome (DGS), who developed generalized dermatitis. The patients shared common T-cell abnormalities, as demonstrated by the low response to mitogenic stimulation, by an unusual usage of specific T-cell receptor (TCR) segments, and by a reduction of TCR diversity in both alpha/beta and gamma/delta populations. Furthermore, they both showed an impaired thymic function, as assessed by the low number of TCR recombination excision circles, which are formed from excised DNA during the rearrangement of TCR genes. These data indicated that generalized erythrodermia may be present in different forms of T-cell immunodeficiency and may reflect intrinsic defects in either V(D)J recombination or in thymic development, leading to the peripheral expansion of T-cell clonotypes, that bear peculiar TCR chains.