Targeting CCR4 with mogamulizumab in refractory CD3-CD4+ lymphocytic-variant hypereosinophilic syndrome.

Not available.

Studying antigen-specific T cells through a streamlined, whole blood-based extracellular approach.

Techniques currently used for the study of antigen-specific T-cell responses are either poorly informative or require a heavy workload. Consequently, many perspectives associated with the broader study of such approaches remain mostly unexplored in translational research. However, these could benefit many fields including but not limited to infectious diseases, oncology, and vaccination. Herein, the main objective of this work was to develop a standardized flow cytometry-based approach that would combine ease of use together with a relevant study of antigen-specific T-cell responses so that they could be more often included in clinical research. To this extent, a streamlined approach relying on 1/ the use of whole blood instead of peripheral blood mononuclear cells and 2/ solely based on the expression of extracellular activation-induced markers (AIMs), called whole blood AIM (WAIM), was developed and further compared to more conventional techniques such as enzyme-linked immunospot (ELISpot) and flow cytometry-based intracellular cytokine staining (ICS). Based on a cohort of 20 individuals receiving the COVID-19 mRNA vaccine and focusing on SARS-CoV-2 and cytomegalovirus (CMV)-derived antigen T-cell-specific responses, a significant level of correlation between the three techniques was found. Based on the use of whole blood and on the expression of extracellular activation-induced markers (CD154, CD137, and CD107a), the WAIM technique appears to be very simple to implement and yet allows interesting patient stratification capabilities as the chosen combination of extracellular markers exhibited higher orthogonality than cytokines that are commonly considered in ICS (IFN-γ, TNF-α, and IL-2).

Serum neutralization of SARS coronavirus 2 Omicron sublineages BA.1 and BA.2 and cellular immune responses 3 months after booster vaccination.

OBJECTIVES: We investigated serum neutralizing activity against BA.1 and BA.2 Omicron sublineages and T cell response before and 3 months after administration of the booster vaccine in healthcare workers (HCWs). METHODS: HCWs aged 18-65 years who were vaccinated and received booster doses of the BNT162b2 vaccine were included. Anti-SARS coronavirus 2 IgG levels and cellular response (through interferon γ ELISpot assay) were evaluated in all participants, and neutralizing antibodies against Delta, BA.1, and BA.2 were evaluated in participants with at least one follow-up visit 1 or 3 months after the administration of the booster dose. RESULTS: Among 118 HCWs who received the booster dose, 102 and 84 participants attended the 1-month and 3-month visits, respectively. Before the booster vaccine dose, a low serum neutralizing activity against Delta, BA.1, and BA.2 was detectable in only 39/102 (38.2%), 8/102 (7.8%), and 12/102 (11.8%) participants, respectively. At 3 months, neutralizing antibodies against Delta, BA.1, and BA.2 were detected in 84/84 (100%), 79/84 (94%), and 77/84 (92%) participants, respectively. Geometric mean titres of neutralizing antibodies against BA.1 and BA.2 were 2.2-fold and 2.8-fold reduced compared with those for Delta. From 1 to 3 months after the administration of the booster dose, participants with a recent history of SARS coronavirus 2 infection (n = 21/84) had persistent levels of S1 reactive specific T cells and neutralizing antibodies against Delta and BA.2 and 2.2-fold increase in neutralizing antibodies against BA.1 (p 0.014). Conversely, neutralizing antibody titres against Delta (2.5-fold decrease, p < 0.0001), BA.1 (1.5-fold, p 0.02), and BA.2 (2-fold, p < 0.0001) declined from 1 to 3 months after the administration of the booster dose in individuals without any recent infection. DISCUSSION: The booster vaccine dose provided significant and similar response against BA.1 and BA.2 Omicron sublineages; however, the immune response declined in the absence of recent infection.

Immunogenicity of BNT162b2 vaccine booster against SARS-CoV-2 Delta and Omicron variants in nursing home residents: A prospective observational study in older adults aged from 68 to 98 years.

Background: The present study aimed to evaluate the persistent immunogenicity offered by a third dose of BNT162b2 against Delta and Omicron variants, in nursing home (NH) residents. Methods: In this monocenter prospective observational study, anti-spike IgG levels, S1 domain reactive T cell counts, serum neutralizing antibody titers against Delta and Omicron variants were compared before and up to three months after the BNT162b2 booster dose, in NH residents without COVID-19 (COVID-19 naive) or with COVID-19 prior to initial vaccination (COVID-19 recovered). Findings: 106 NH residents (median [interquartile range] age: 86·5 [81;91] years) were included. The booster dose induced a high increase of anti-spike antibody levels in all subjects (p < 0.0001) and a mild transient increase of specific T cells. Before the booster dose, Delta neutralization was detected in 19% (n = 8/43) and 88% (n = 37/42) of COVID-19 naive and COVID-19 recovered subjects, respectively. Three months after the booster dose, all NH residents developed and maintained a higher Delta neutralization (p < 0·0001). Before the booster dose, Omicron neutralization was detected in 5% (n = 2/43) and 55% (n = 23/42) of COVID-19 naive and COVID-19 recovered subjects, respectively, and three months after, in 84% and 95%, respectively. Neutralizing titers to Omicron were lower than to Delta in both groups with a 35-fold reduction compared to Delta. Interpretation: The booster dose restores high neutralization titers against Delta in all NH residents, and at a lower level against Omicron in a large majority of participants. Future studies are warranted to assess if repeated BNT162b2 booster doses or new specific vaccines might be considered for protecting such fragile patients against Omicron and/or future SARS-CoV-2 variants. Funding: French government through the Programme Investissement d'Avenir (I-SITE ULNE/ANR-16-IDEX-0004 ULNE) and the Label of COVID-19 National Research Priority (National Steering Committee on Therapeutic Trials and Other COVID-19 Research, CAPNET).

Impaired Functional T-Cell Response to SARS-CoV-2 After Two Doses of BNT162b2 mRNA Vaccine in Older People.

Long-term care facility (LTCF) older residents display physiological alterations of cellular and humoral immunity that affect vaccine responses. Preliminary reports suggested a low early postvaccination antibody response against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The aim of this study was to focus on the specific T-cell response. We quantified S1-specific IgG, neutralizing antibody titers, total specific IFNγ-secreting T cells by ELISpot, and functionality of CD4+- and CD8+-specific T cells by flow cytometry, after two doses of the BNT162b2 vaccine in younger and older people, with and without previous COVID-19 infection (hereafter referred to as COVID-19-recovered and COVID-19-naive subjects, respectively). Frailty, nutritional, and immunosenescence parameters were collected at baseline in COVID-19-naive older people. We analyzed the immune response in 129 young adults (median age 44.0 years) and 105 older residents living in a LCTF (median age 86.5 years), 3 months after the first injection. Humoral and cellular memory responses were dramatically impaired in the COVID-19-naive older (n = 54) compared with the COVID-19-naive younger adults (n = 121). Notably, older participants' neutralizing antibodies were 10 times lower than the younger's antibody titers (p < 0.0001) and LCTF residents also had an impaired functional T-cell response: the frequencies of IFNγ+ and IFNγ+IL-2+TNFα+ cells among specific CD4+ T cells, and the frequency of specific CD8+ T cells were lower in COVID-19-naive older participants than in COVID-19-naive young adults (p < 0.0001 and p = 0.0018, respectively). However, COVID-19-recovered older participants (n = 51) had greater antibody and T-cell responses, including IFNγ+ and IFNγ+IL-2+TNFα+-specific CD4+ T cells (p < 0.0001), as well as TNFα+-specific CD8+ T cells (p < 0.001), than COVID-19-naive older adults. We also observed that "inflammageing" and particularly high plasma levels of TNFα was associated to poor antibody response in the older participants. In conclusion, our results show that the COVID-19-naive older people had low counts and impaired specific CD4+ and CD8+ T cells, in addition to impaired antibody response, and that specific studies are warranted to assess the efficiency of SARS-CoV-2 mRNA-based vaccines, as in other immunocompromised subjects. Our study also shows that, despite their physiological alterations of immunity, vaccination is highly efficient in boosting the prior natural memory response in COVID-19-recovered older people.

Monitoring CAR T-cells using flow cytometry.

BACKGROUND: Chimeric antigen receptor (CAR) T-cell therapy is considered as a major scientific breakthrough in cancer immunotherapy. The success of adoptive CAR T-cell therapy for cancer has inspired researchers to expand indications into the area of solid tumors, autoimmune and infectious diseases. The most important factors influencing outcome and durability of the response after infusion of CAR T-cell are proliferation and persistence of this cell subset. It becomes therefore important to detect easily and monitor circulating CAR T-cells into blood samples. Approaches such as quantitative PCR (qPCR) or flow cytometry have been developed. The aim of this study was to set up and optimize a reachable flow cytometry technique using labeled CD19 protein for the measurement of CAR T-cells in infusion bag and patient's blood. METHODS: Patients receiving Yescarta in Cell Therapy Unit (Department of hematology, Lille university hospital, France) between April and October 2019 and healthy volunteers were included to set up the flow cytometry technique. RESULTS AND CONCLUSIONS: We assessed feasibility in clinic and suitability to routine workload of a flow cytometry technique to follow CAR T-cells in infusion bag and patient's blood. With only a few manual steps, the present protocol allows the technician to perform this technique among other routine tasks, meaning a time to results of <2 hr after sample reception. We were also able to assess CAR T-cell heterogenity in terms of CD4+ and CD8+ T lymphocytes within the subset. Moreover, this technique allows monitoring of both authority approved CD19 CAR T-cell.

Severe SARS-CoV-2 patients develop a higher specific T-cell response.

OBJECTIVES: Assessment of the adaptive immune response against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is crucial for studying long-term immunity and vaccine strategies. We quantified IFNγ-secreting T cells reactive against the main viral SARS-CoV-2 antigens using a standardised enzyme-linked immunospot assay (ELISpot). METHODS: Overlapping peptide pools built from the sequences of M, N and S viral proteins and a mix (MNS) were used as antigens. Using IFNγ T-CoV-Spot assay, we assessed T-cell and antibody responses in mild, moderate and severe SARS-CoV-2 patients and in control samples collected before the outbreak. RESULTS: Specific T cells were assessed in 60 consecutive patients (mild, n = 26; moderate, n = 10; and severe patients, n = 24) during their follow-up (median time from symptom onset [interquartile range]: 36 days [28;53]). T cells against M, N and S peptide pools were detected in n = 60 (100%), n = 56 (93.3%), n = 55 patients (91.7%), respectively. Using the MNS mix, IFNγ T-CoV-Spot assay showed a specificity of 96.7% (95% CI, 88.5-99.6%) and a specificity of 90.3% (75.2-98.0%). The frequency of reactive T cells observed with M, S and MNS mix pools correlated with severity and with levels of anti-S1 and anti-RBD serum antibodies. CONCLUSION: IFNγ T-CoV-Spot assay is a reliable method to explore specific T cells in large cohorts of patients. This test may become a useful tool to assess the long-lived memory T-cell response after vaccination. Our study demonstrates that SARS-CoV-2 patients developing a severe disease achieve a higher adaptive immune response.

Pancreatic beta cells persistently infected with coxsackievirus B4 are targets of NK cell-mediated cytolytic activity.

It has been suggested that the persistence of coxsackieviruses-B (CV-B) in pancreatic beta cells plays a role in the pathogenesis of type 1 diabetes (T1D). Yet, immunological effectors, especially natural killer (NK) cells, are supposed to clear virus-infected cells. Therefore, an evaluation of the response of NK cells to pancreatic beta cells persistently infected with CV-B4 was conducted. A persistent CV-B4 infection was established in 1.1B4 pancreatic beta cells. Infectious particles were found in supernatants throughout the culture period. The proportion of cells containing viral protein VP1 was low (< 5%), although a large proportion of cells harbored viral RNA (around 50%), whilst cell viability was preserved. HLA class I cell surface expression was downregulated in persistently infected cultures, but HLA class I mRNA levels were unchanged in comparison with mock-infected cells. The cytolytic activities of IL-2-activated non-adherent peripheral blood mononuclear cells (PBMCs) and of NK cells were higher towards persistently infected cells than towards mock-infected cells, as assessed by an LDH release assay. Impaired cytolytic activity of IL-2-activated non-adherent PBMCs from patients with T1D towards infected beta cells was observed. In conclusion, pancreatic beta cells persistently infected with CV-B4 can be lysed by NK cells, implying that impaired cytolytic activity of these effector cells may play a role in the persistence of CV-B in the host and thus in the viral pathogenesis of T1D.

Donor-derived CD4+/CCR7+ T-cell impact on acute GVHD incidence following haplo-HCT after reduced intensity conditioning and posttransplant cyclophosphamide.

In previous studies, we and others observed in patients undergoing HLA-matched hematopoietic cell transplantation that high proportion of donor-derived CD4+/CCR7+ T cells were associated with an increased risk of acute GVHD without any interference in relapse incidence. We investigated the impact of donor-derived CD4+/CCR7+ T cells on patient outcome in haploidentical settings where posttransplant cyclophosphamide is used. We analyzed T-cell subsets in grafts of 29 adult patients who underwent first haploidentical transplant following reduced intensity conditioning. The median CD4+/CCR7+ subset proportion was 69.2% among donor CD4+ T cells. With a median follow-up of 28.1 months (range: 11.0-44.3), 16 patients (55%) developed acute GVHD; this includes 5 patients with grade 3 acute GVHD. Fifty-four percent of patients who received > 69.2% of CD4+/CCR7+ T cells and 12% of patients who received < 69.2% CD4+/CCR7+ T cells developed acute GVHD (p = 0.028). In multivariate analysis, a high proportion of CD4+/CCR7+ T cells was the only factor that impacted acute GVHD (HR = 4.925, 95% CI [1.020-23.775], p = 0,047) with no impact on overall survival. Our results confirm the impact of a high proportion of CD4+/CCR7+ T cells on acute GVHD incidence in patients undergoing haploidentical transplant despite the use of posttransplant cyclophosphamide.

A standardized flow cytometry procedure for the monitoring of regulatory T cells in clinical trials.

BACKGROUND: Quantification of regulatory T cells (Tregs) is crucial in immunomonitoring in clinical trials as this cell population has been shown to be involved in a wide range of diseases, including cancers, autoimmune diseases, infections, and allergies. Human Tregs are defined as CD4+ CD25+ CD127low FoxP3+ cells, and the standardization of their staining by flow cytometry is a challenge, especially in multicenter clinical trials, notably because of the intracellular location of FoxP3. METHOD: A flow cytometry staining procedure was settled and standardized to measure human Tregs in peripheral whole blood using precoated dried antibodies in ready-to-use tubes. It was compared with reference methods and implemented and validated to be suitable with different cytometer platforms. RESULTS: The standardized protocol developed with dried antibodies and reduced volumes of whole blood allows an optimal identification of Tregs. Compared with classical staining procedure, it reduces the number of steps required, in a very fast and simple technique. The accuracy of the method was confirmed by a multicenter comparison with different cytometer brands. CONCLUSIONS: Our results highlight the reliability of this high-standard protocol that could become a reference method for the monitoring of Tregs in clinical trials. © 2018 International Clinical Cytometry Society.

Coxsackievirus B4 Can Infect Human Peripheral Blood-Derived Macrophages.

Beyond acute infections, group B coxsackieviruses (CVB) are also reported to play a role in the development of chronic diseases, like type 1 diabetes. The viral pathogenesis mainly relies on the interplay between the viruses and innate immune response in genetically-susceptible individuals. We investigated the interaction between CVB4 and macrophages considered as major players in immune response. Monocyte-derived macrophages (MDM) generated with either M-CSF or GM-CSF were inoculated with CVB4, and infection, inflammation, viral replication and persistence were assessed. M-CSF-induced MDM, but not GM-CSF-induced MDM, can be infected by CVB4. In addition, enhancing serum was not needed to infect MDM in contrast with parental monocytes. The expression of viral receptor (CAR) mRNA was similar in both M-CSF and GM-CSF MDM. CVB4 induced high levels of pro-inflammatory cytokines (IL-6 and TNFα) in both MDM populations. CVB4 effectively replicated and persisted in M-CSF MDM, but IFNα was produced in the early phase of infection only. Our results demonstrate that CVB4 can replicate and persist in MDM. Further investigations are required to determine whether the interaction between the virus and MDM plays a role in the pathogenesis of CVB-induced chronic diseases.

CD3-CD4+ lymphoid variant of hypereosinophilic syndrome: nodal and extranodal histopathological and immunophenotypic features of a peripheral indolent clonal T-cell lymphoproliferative disorder.

The CD3(-)CD4(+) lymphoid variant of hypereosinophilic syndrome is characterized by hypereosinophilia and clonal circulating CD3(-)CD4(+) T cells. Peripheral T-cell lymphoma has been described during this disease course, and we observed in our cohort of 23 patients 2 cases of angio-immunoblastic T-cell lymphoma. We focus here on histopathological (n=12 patients) and immunophenotypic (n=15) characteristics of CD3(-)CD4(+) lymphoid variant of hypereosinophilic syndrome. Atypical CD4(+) T cells lymphoid infiltrates were found in 10 of 12 CD3(-)CD4(+) L-HES patients, in lymph nodes (n=4 of 4 patients), in skin (n=9 of 9) and other extra-nodal tissues (gut, lacrymal gland, synovium). Lymph nodes displayed infiltrates limited to the interfollicular areas or even an effacement of nodal architecture, associated with proliferation of arborizing high endothelial venules and increased follicular dendritic cell meshwork. Analysis of 2 fresh skin samples confirmed the presence of CD3(-)CD4(+) T cells. Clonal T cells were detected in at least one tissue in 8 patients, including lymph nodes (n=4 of 4): the same clonal T cells were detected in blood and in at least one biopsy, with a maximum delay of 23 years between samples. In the majority of cases, circulating CD3(-)CD4(+) T cells were CD2(hi) (n=9 of 14), CD5(hi) (n=12 of 14), and CD7(-)(n=4 of 14) or CD7(low) (n=10 of 14). Angio-immunoblastic T-cell lymphoma can also present with CD3(-)CD4(+) T cells; despite other common histopathological and immunophenotypic features, CD10 expression and follicular helper T-cell markers were not detected in lymphoid variant of hypereosinophilic syndrome patients, except in both patients who developed angio-immunoblastic T-cell lymphoma, and only at T-cell lymphoma diagnosis. Taken together, persistence of tissular clonal T cells and histopathological features define CD3(-)CD4(+) lymphoid variant of hypereosinophilic syndrome as a peripheral indolent clonal T-cell lymphoproliferative disorder, which should not be confused with angio-immunoblastic T-cell lymphoma.

A low effective dose of interleukin-7 is sufficient to maintain cord blood T cells alive without potentiating allo-immune responses.

Slow reconstitution of T cell immunity remains a critical issue after umbilical cord blood (CB) transplantation. Although this may be a consequence of the low cell dose, it may also reflect the propensity of naïve T cells, which predominate in CB, to undergo apoptotic cell death. Exogenous interleukin 7 (IL-7) can prevent apoptosis of naïve T cells, but at high concentrations, IL-7 may also expand alloreactive T cells, thereby aggravating the risk of graft-versus-host disease. We evaluated the survival of CB T cells from 34 healthy full-term pregnancies, and we found wide interdonor variation, from 17.4% to 79.7%, of CB T cells that were still alive after being rested for 4 days in culture medium without cytokine supplementation. The viability of CB T cells was negatively correlated to infant birth weight (Spearman's ρ = .376; P = .031) and positively correlated to venous CB pH (ρ = .397; P = .027); both associations were confirmed by multivariate analysis (P = .023 and P = .005, respectively). A low supplemental concentration (100 pg/mL) of recombinant human IL-7 was sufficient to maintain the viability of cryopreserved/thawed CB T cells, with most (>80%) cells remaining in a quiescent state and without significant changes in their CD4/CD8 ratio and the proportion of CD4(+) CD31(+) PTK7(+) recent thymic emigrants. IL-7 at 100 pg/mL did not lead to any significant enhancement of the alloreactive response of CB T cells, as evaluated by proliferation rates (thymidine incorporation and carboxyfluorescein diacetate succinimidyl ester dilution) and interferon-gamma production (ELISPOT). This effective concentration of IL-7 is far lower than that obtained in vivo after pharmacological administration of the cytokine. This study suggests that administration of lower doses of recombinant human IL-7 than used in previous clinical trials may be sufficient to sustain the viability of infused CB T cells and, thus, help to accelerate naïve T cell reconstitution without potentiating their alloreactivity.

The lymphoid variant of hypereosinophilic syndrome: study of 21 patients with CD3-CD4+ aberrant T-cell phenotype.

The CD3-CD4+ aberrant T-cell phenotype is the most described in the lymphoid variant of hypereosinophilic syndrome (L-HES), a rare form of HES. Only a few cases have been reported, and data for these patients are scarce. To describe characteristics and outcome of CD3-CD4+ L-HES patients, we conducted a national multicentric retrospective study in the French Eosinophil Network. All patients who met the recent criteria of hypereosinophilia (HE) or HES and who had a persistent CD3-CD4+ T-cell subset on blood T-cell phenotyping were included. Clinical and laboratory data were retrospectively collected by chart review. CD3-CD4+ L-HES was diagnosed in 21 patients (13 females, median age 42 years [range, 5-75 yr]). Half (48%) had a history of atopic manifestations. Clinical manifestations were dermatologic (81%), superficial adenopathy (62%), rheumatologic (29%), gastrointestinal (24%), pulmonary (19%), neurologic (10%), and cardiovascular (5%). The median absolute CD3-CD4+ T-cell count was 0.35 G/L (range, 0.01-28.3), with a clonal TCRγδ rearrangement in 76% of patients. The mean follow-up duration after HES diagnosis was 6.9 ± 5.1 years. All patients treated with oral corticosteroids (CS) (n = 18) obtained remission, but 16 required CS-sparing treatments. One patient had a T-cell lymphoma 8 years after diagnosis, and 3 deaths occurred during follow-up.In conclusion, clinical manifestations related to CD3-CD4+ T cell-associated L-HES are not limited to skin, and can involve all tissue or organs affected in other types of HE. Contrary to FIP1L1-PDGFRA chronic eosinophilic leukemia patients, CS are always effective in these patients, but CS-sparing treatments are frequently needed. The occurrence of T-cell lymphoma, although rare in our cohort, remains a major concern during follow-up.

Naïve subset develops the most important alloreactive response among human CD4+ T lymphocytes in human leukocyte antigen-identical related setting.

In longitudinal clinical studies, receiving a high percentage of allogeneic donor-derived CD4(+) CCR7(+) T cells, which include naïve and central memory subsets have been correlated with increased incidence and severity of acute GVHD. Whether naïve and central memory CD4(+) T-cell subsets contribute more or equally to alloimmune responses are still unclear in human. The aim of this study was to investigate in vitro the alloreactive response of purified naïve, central memory, and effector memory CD4(+) T-cell subsets in HLA identical setting. By coculturing monocyte-derived dendritic cells and purified CD4(+) T-cell subsets, from healthy HLA-identical male and female sibling pairs, we found that naïve CD4(+) CCR7(+) CD45RA(+) T cells developed the highest proliferative response upon stimulation by minor histocompatibility antigens and were progressively driven to produce high levels of interferon-γ, tumor necrosis factor, and interleukin-6. Comparatively, the central memory CD4(+) CCR7(+) CD45RA(neg) subset proliferated to a lower extent and produced very low amounts of pro-inflammatory cytokines while the CCR7(neg) effector memory CD4(+) subset was unresponsive. This study demonstrates the superior capacity of naïve CD4(+) T cells to mount a primary alloreactive response as compared to central memory T cells. Their proliferative response associated with a pro-inflammatory differentiation makes them potentially acute GVHD inducers. These in vitro results in line with what we have observed in clinical studies and may also lend support to approaches of partial selective T-cell depletion for GVHD prevention.

Defect in recruiting effector memory CD8+ T-cells in malignant pleural effusions compared to normal pleural fluid.

BACKGROUND: Malignant pleural effusions (MPE) are a common and fatal complication in cancers including lung or breast cancers, or malignant pleural mesothelioma (MPM). MPE animal models and immunotherapy trials in MPM patients previously suggested defects of the cellular immunity in MPE. However only few observational studies of the immune response were done in MPM patients, using questionable control groups (transudate…). METHODS: We compared T cell populations evaluated by flow cytometry from blood and pleural effusion of untreated patients with MPM (n = 58), pleural metastasis of adenocarcinoma (n = 30) or with benign pleural lesions associated with asbestos exposure (n = 23). Blood and pleural fluid were also obtained from healthy subjects, providing normal values for T cell populations. RESULTS: Blood CD4+ or CD8+ T cells percentages were similar in all groups of patients or healthy subjects. Whereas pleural fluid from healthy controls contained mainly CD8+ T cells, benign or malignant pleural effusions included mainly CD4+ T cells. Effector memory T cells were the main T cell subpopulation in pleural fluid from healthy subjects. In contrast, there was a striking and selective recruitment of central memory CD4+ T cells in MPE, but not of effector cells CD8+ T cells or NK cells in the pleural fluid as one would expect in order to obtain an efficient immune response. CONCLUSIONS: Comparing for the first time MPE to pleural fluid from healthy subjects, we found a local defect in recruiting effector CD8+ T cells, which may be involved in the escape of tumor cells from immune response. Further studies are needed to characterize which subtypes of effector CD8+ T cells are involved, opening prospects for cell therapy in MPE and MPM.

Primed status of transitional B cells associated with their presence in the cerebrospinal fluid in early phases of multiple sclerosis.

In the present study we showed that transitional B cells of patients with clinically isolated syndrome (CIS) and relapsing-remitting multiple sclerosis (RR-MS) are reduced in the peripheral blood (PB) (5.5- and 3.7-fold, respectively). In addition, these cells appeared to up-regulate different integrins (α4 and ß1). These observations were associated with a primed cellular status, confirmed by an increased proportion of circulating CD80(+) transitional B cells. Interestingly, these results correlate with presence of transitional B cells in the CSF. Furthermore, these cells were absent in the CSF of individuals with other inflammatory neurological disease, and their levels in paired PB and CD80 expression were normal. Altogether, our data revealed that a differential primed status of transitional B cells is a characteristic feature of early phases of MS disease, and this functional status is associated with the ability of these cells to cross the blood-CSF barrier.

B-cell subsets up-regulate α4 integrin and accumulate in the cerebrospinal fluid in clinically isolated syndrome suggestive of multiple sclerosis onset.

During the pathogenesis of multiple sclerosis (MS), activated B-cells cross the inflamed endothelium of the central nervous system (CNS) to exert their effector functions, probably associated with the action of several adhesion molecules. B-cell mobilization towards the CNS already occurs after the first demyelinating events suggestive of MS, known as clinically isolated syndrome (CIS). However, little is known about the role of these adhesion molecules at this early disease stage. We, therefore, evaluated the relationship between the expression of α4 integrin by peripheral B-cell subsets and disease activity. We found that α4 integrin was up-regulated in all B-cell subsets from patients with CIS and that its expression was correlated with the number of gadolinium-enhanced lesions. A comparison of B-cell subsets distribution in the CSF of patients with CIS, of patients with other inflammatory neurological diseases (OIND) and of patients with non-inflammatory neurological diseases (NIND) showed that the percentages of CSF CD19(+) B-cells were significantly higher in the CIS and OIND group. In addition, CIS and OIND CSF were enriched in switched and MZ-like memory B-cells, although all B-cell subsets were present. These results suggest that up-regulation of α4 integrin may enhance B-cell accumulation within the CSF at the time of CIS.

Using human CD20-transfected murine lymphomatous B cells to evaluate the efficacy of intravitreal and intracerebral rituximab injections in mice.

PURPOSE: The treatment of primary central nervous system lymphoma (PCNSL) and its subset, primary intraocular lymphoma (PIOL), remains of limited efficiency, and salvage therapies are often used without prior testing in adequate animal models. Most PNCSL/PIOL are aggressive B-cell malignancies. Two animal models that closely mimic the human situation were established to evaluate the efficiency of intravitreal and intracerebral anti-CD20 monoclonal antibody (rituximab) injections. METHODS: Human CD20-transfected murine B-lymphoma cells (38C13 CD20(+)) were inoculated in the vitreous through the pars plana or in the caudate nucleus with the use of a stereotaxic frame in immunocompetent syngeneic mice. Animals were monitored clinically and by funduscopic and histologic examination. Rituximab was injected intravitreally or intracerebrally. Occurrences of exophthalmia, neurologic disturbance, and weight loss were monitored over 2 months. RESULTS: Inoculation of 38C13 CD20(+) cells in the eye or the brain resulted in tumor occurrence after a median of 15 days or 22 days, respectively, with histologic characteristics closely resembling those of PIOL and PCNSL. Local rituximab injections eradicated tumor colonization in more than half the graft recipients and inhibited tumor progression significantly in the others compared with progression in mice that underwent grafting with the control 38C13 cell line (no human CD20 expression) and in mice that underwent grafting with 38C13 CD20(+) cells that received local injections of an irrelevant antibody (trastuzumab). CONCLUSIONS: Inoculation of native or human CD20-transfected murine 38C13 cells in the vitreous or the brain of immunocompetent mice provides useful novel models for evaluating the biology and treatment of PIOL and PCNSL. Intravitreal and intracerebral rituximab injections reduced tumor occurrence and growth in each model.