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.

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.

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.

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.

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.

Selective depletion in HIV infection of T cells that bear specific T cell receptor V beta sequences.

The mechanism of T cell depletion during infection with the human immunodeficiency virus (HIV) is unclear. Examination of the repertoire of T cell receptor V (variable) regions in persons infected with HIV revealed the absence of a common set of V beta regions, whereas V alpha usage was normal. The lack of these V beta segments did not appear to correlate with opportunistic infections. The selective elimination of T cells that express a defined set of V beta sequences may indicate the presence of an HIV-encoded superantigen, similar to those encoded by the long terminal repeat of the mouse mammary tumor virus.

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.

T cell repertoire and autoimmune diseases.

Self-reactivity and autoimmunity are processes related to the breakage of self-tolerance that can be distinguished by their different clinical outcome and are widely accepted cornerstones of immunology. The finding that several potentially autoaggressive cells contribute to the repertoire of healthy individuals has stimulated a great deal of experimental work aimed at understanding the mechanisms that prevent autoimmune pathology. In this review we will consider the basic principles, and our present knowledge of the rules that preside over the interplay of the immune system with self-components. One viewpoint stresses the importance of major histocompatibility complex (MHC) and non-MHC genes in determining genetic predisposition to develop autoimmune phenomena. At a different level there is a strong interest in understanding the mechanisms of processing and presentation of self antigens, especially during ontogeny. Another topic of major interest concerns the interaction between MHC genes and the T cell receptor (TcR) complex as well as the identification of TcR V genes that are preferentially expressed by autoimmune T cells. All of these aspects are evaluated in the context of tolerance based on deletion and anergy. Finally we will propose a general model of autoimmunity based on the most recent findings concerning the biological activity of exogenous superantigens.

Analysis of amplified T cell receptor V beta transcripts by a non-isotopic immunoassay.

We have recently described a new colorimetric DNA enzyme immunoassay (DEIA) for detecting specific hybrids of complementary nucleic acids. This technology is based on an antibody that selectively recognizes double, but not single stranded DNA and therefore reveals the hybridization event independently from the DNA sequences. Most importantly, the test has an ELISA format and is very rapid and convenient for processing large numbers of samples. In the present report we have adapted this method to reveal the specificity of amplified T cell receptor V beta transcripts. V beta genes were amplified by polymerase chain reaction, using family specific primers and the specificity of the amplified products was determined by Southern blot and by DEIA. Our data demonstrate that DEIA had the same degree of sensitivity and specificity of conventional Southern hybridization. The possibility of analyzing amplified products with the simplicity of a conventional immunoassay should greatly facilitate the analysis of complex multigenic systems such as the T cell receptor and the immunoglobulin repertoire.

Detection of skewed T-cell receptor V-beta gene usage in the peripheral blood of patients with multiple sclerosis.

The ex vivo analysis of the T-cell receptor V-beta (TCRBV) gene usage by circulating T lymphocytes in Multiple Sclerosis (MS) patients may contribute to understanding disease pathogenesis. In the present study, TCRBV gene usage was analyzed in freshly collected unstimulated peripheral blood mononuclear cells (PBMC) isolated from 40 MS patients and 20 healthy controls. Nine patients presented abnormal repertoires, with expansion of one or more TCRBV segments. Among these patients, six presented expansion of TCRBV9 chain expression, three also having an expansion of TCRBV1, TCRBV11 and TCRBV22 segments. The most frequently observed TCRBV chain expansion, TCRBV9, was further analyzed and identified as polyclonal. Evaluation of clinical variables showed that median disease duration was shorter in patients with TCRBV gene expression abnormalities. Longitudinal evaluation of five patients with a skewed repertoire showed regression of expanded TCRBV chains expression to normal values. These data indicate that certain MS patients have abnormal TCRBV gene expression. Such abnormalities are caused by polyclonal expansions of T lymphocyte subpopulations that use the same TCRBV gene families, are unstable and preferentially observed early in the course of the disease.

Assessment of T-cell receptor beta-chain diversity by heteroduplex analysis.

The aim of this work was to search for a simple and alternative approach to the currently used methodologies for the analysis of T-cell receptor repertoire diversity. To this end we studied whether the heteroduplex analysis could be adapted to study the clonality of the T-cell receptor beta chain (TCRBV). We therefore analyzed, by sequencing, the molecular characteristics of the V-D-J junctions of numerous TCRBV chains from a variety of patients and from normal individuals, and compared the results with those obtained with the heteroduplex analysis. The latter procedure involves the amplification of the target TCRBV chains and the denaturation and renaturation of the amplified product to permit the random association of the distinct DNA strands encoding the different junctional regions. Whereas amplified material from polyclonal lymphoid cells migrates on a polyacrylamide gel as a "smear" of bands composed of different-sized polyclonal PCR fragments, the mismatched chains derived from oligoclonal populations migrate as discrete "heteroduplexes" and can be separated from the matched "homoduplex" obtained from homogeneous clonal cells. Our results provide evidence demonstrating that heteroduplex analysis can successfully be applied to the analysis of T-cell clonality in a variety of samples and can be complementary or substitute for the standard approach of TCR cloning and multiple sequencing of junctional regions. Thus, the procedure should facilitate the implementation of the analysis of TCR in diagnostic routine and should find applications in numerous physiologic and pathologic conditions.

Non-radioisotopic methods for DNA probes.

We have recently developed a new colorimetric method, DNA enzyme immunoassay (DEIA), for detecting specific hybrids of complementary nucleic acids. This technology is based on an antibody that selectively recognizes double, but not single-stranded DNA. The molecule does not react with a specific probe immobilized on microwells through an avidin-biotin bridge, nor with non-specific amplified sequences, since they are removed by washing. The antibody reveals the hybridization event, independently of the DNA sequences and, for this reason, the method is broadly applicable and extremely versatile. Although we chose a format based on the immobilization of the probe through an avidin-biotin interaction, DEIA assay can also be applied to other analytical schemes that do not require any modification of the probe. Most importantly, the test has an ELISA format and is rapid and convenient for processing large numbers of samples. This technology has been applied, in our laboratory, to different areas, including virology, genetic and basic immunology. The DEIA assay has been successfully used to detect the presence of hepatitis B (HBV), hepatitis C (HCV) and hepatitis delta virus (HDV) sequences in serum of patients, to discriminate different HLA alleles, to identify mutations in the Cystic Fibrosis gene, and to investigate the role of the T cell receptor in some immunological diseases. The results obtained in all our experiments demonstrated that the advantages offered by the assay do not penalize its analytical performance as compared to conventional Southern blot.

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.

Expression and combinatorial diversity of germ line-encoded T cell receptor V genes in human peripheral blood T cells.

The potential diversity of the T cell receptor (TcR) is defined by the combinational expression of variable segments and by mechanisms that insert or delete nucleotides at the junctional regions. The available repertoire is strongly influenced by negative and positive selection events. To study whether the diversity of the human T cell receptor of peripheral T cells is further restricted by the interaction between the TcR alpha and beta chains, we compared the level of transcription of different V alpha elements in human T cell blasts expressing either restricted or unrestricted sets of V beta genes. Our data establish that in some individuals, but not in others, the transcription of a given V alpha element is independent from the presence of particular V beta transcripts. Furthermore, our data also suggest that, in contrast to mouse, major TcR V gene deletions are absent in humans. Taken collectively, these results indicate that the diversity of the peripheral human TcR repertoire can benefit from the combinatorial expression of all the V elements present in the genome.

Use of variable human V delta genes to create functional T cell receptor alpha chain transcripts.

Previous studies of the human T cell receptor delta genes identified five commonly used V delta segments distinct from any of the known V alpha genes. To define better the relationship between the T cell receptor delta and alpha repertoires we amplified cDNA obtained by polyclonally activated lymphocytes with a common 3' antisense C alpha-specific primer and with five different 5' sense V delta family-specific primers. Amplified products were detected in staphylococcal enterotoxin C2, staphylococcal enterotoxin E, phytohemagglutinin, concanavalin A, anti-CD3 and anti-V beta 8-activated cells, although each cell population expressed a selective pattern of V delta genes. Sequence analysis revealed that each of the known V delta genes can productively rearrange to J alpha segments to produce functional V delta-J alpha-C alpha transcripts. These results argue strongly against the notion that the human V delta and V alpha repertoires are distinct. They further suggest that the restricted delta repertoire observed in many gamma/delta clones results from selection rather than from controlled rearrangements at the T cell receptor alpha/delta locus.

Expression of the human V beta 8 gene product preferentially correlates with class II major histocompatibility complex restriction specificity.

We report that, in peripheral blood T cells, the V beta 8 gene product is much more represented on CD4+CD8- than on CD4-CD8+ lymphocytes. This skewing was observed in all individuals tested and with two independent strategies, one of which allows the selection of V beta 8+ T cells independently from their major histocompatibility complex-antigen specificity. Our data imply that the human V beta 8 gene product confers class II restriction specificity and that both the T cell receptor chains and CD4 molecule are involved in the selection of the T cell repertoire.

Positive and negative immunoregulation through CD4 depends on the concentration of the specific ligand and on the state of activation of the responding cells.

In order to better define the functional role of the interaction of CD4 polypeptide with the T-cell receptor (TCR) complex, we analysed the effect of an anti-CD4 monoclonal antibody (mAb) on T-cell activation and on the modulation of expression of CD3, CD4 and TCR variable (V) regions. The results presented here demonstrate that both positive and negative modulation of CD3 and TCR V regions can be obtained with different concentrations of the same anti-CD4 mAb and that these effects are functionally directly related to differences in IL2-receptor expression. Moreover, our data show that the dose of anti-CD4 mAb required for modulating CD3- and CD4-molecule expression on activated E+ peripheral blood mononuclear cells is at least 30 times higher than that required to obtain the same effect on resting cells. Thus, our results demonstrate that the interaction of CD4 molecule with its ligand can result in both up and downregulation of TCR and IL2-receptor expression, and that this differential modulation is strictly dependent on the concentration of available ligand as well as on the activation state of the responding cells.