Thymus-independent T-cell differentiation in vitro

The generatation of large quantities of novel human T-cell clones ex vivo would make a wide range of gene- and immuno-therapies for tumours and viral infections possible. Several techniques have been described to generate, in vitro and in vivo (using xenogenic hosts), mature T cells from fetal-neonatal and adult human CD34⁺ cells. All these techniques are cumbersome and cannot be easily translated into clinical protocols because they involve co-cultivation of CD34⁺ cells with thymic fragments from either human or murine fetuses. We report that the mononuclear cells of human cord blood contain a cell population that supports the differentiation of CD34⁺ cells into CD4⁺ or CD8⁺ naive T cells in serum-deprived cultures stimulated with stem cell factor and interleukin 7. CD4⁺ or CD8⁺ CD45RA⁺ TCRαβ⁺ T cells were continuously produced in vitro over a period of 20 d under these conditions. The generation of T cells in these cultures was a dynamic process and clones of T cells expressing new T-cell receptor β-chain rearrangments were generated over time. These results pave the way for the development of very simple culture conditions for ex-vivo production of naive helper or cytotoxic T cells which could be very useful for gene- and immuno-therapy of human diseases.     

A biclonal large granular lymphocyte (LGL)/NK-associated (NKa) disorder of CD4+ and CD8+ lymphocyte subpopulations characterized by the simultaneous presence of distinct TCR rearrangements

Summary. This communication reports a patient with concomitant expansions of CD4⁺ and CD8⁺ large granular lymphocytes. Immunological analyses revealed that the abnomally increased CD4⁺ LGL fraction was phenotypically similar to other reported persistent CD4⁺ LGL expansions, whereas the phenotypic profile for the CD8⁺ LGL component was unusual. of particular note was the finding that both the CD4⁺ and CD8⁺ LGL fractions showed high membrane the CD45RO isoform expression, thus suggesting their 'primed' status. Molecular biology studies of immunomagnetically fractionated cells using a Tγ9 TCR gamma gene primer further revealed that the CD4⁺ and CD8⁺ components were both clonal but showed different patterns of rearrangement It is suggested that the simultaneous presence of CD4⁺ and CD8⁺ clonal populations are unlikely to have been derived from a common progenitor and that they reflect expansions of functionally restricted subpopulations     

T-cell form of chronic lymphocytic leukaemia: a reaffirmation of its existence

Early in the 1980s three categories of T-cell chronic lymphocytic leukaemia were recognized: CD4⁺ CD8⁻ knobby type, CD4⁻ CD8⁺ azurophilic type and CD4⁺ CD8⁻ adult T-cell leukaemia (ATL) type. Both azurophilic and ATL types were later shown to be distinctive disorders, whereas the knobby type has been largely neglected and even considered non-existent by some authors. In this report we describe two patients with leukaemia of CD3⁺ CD4⁺ CD8⁻ post-thymic T lymphocytes presenting with marked lymphocytosis, generalized lymphadenopathy and hepatosplenomegaly. We believe that CLL of post-thymic T-lymphocytes is a distinct entity, and merits a separate designation from other T-cell leukaemias.     

New Approaches to the Management of the Wolff-Parkinson-White Syndrome

Summary: New approaches to the management of the Wolff-Parkinson-White syndrome.
Pre-excitation has been defined as "a condition in which all or some portion of the ventricular muscle is activated earlier in relation to atrial events, than would be expected had the impulse reached the ventricle by way of the normal atrioventricular conduction system".'¹ The susceptibility of patients with pre-excitation to paroxysmal tachycardias had been described many years previously.² The Wolff-Parkinson-White syndrome was initially thought to have a relatively benign prognosis. Subsequently, it was recognised that some patients were prone to life-threatening arrhythmias and sudden death.^{3, 4} This recognition has given impetus to sophisticated electrophysiological study of the pre-excitation syndromes by recently developed techniques of catheterization^{5 6}'⁷, epicardial mapping⁸ and surgery.³ The purpose of this review is to highlight the recent advances in management of the Wolff-Parkinson-White syndrome which have been based upon these electrophysiological techniques.     

CD3+ CD8+ CD56− clonal large granular lymphocyte leukaemia and HIV infection

High-grade malignant lymphomas associated with HIV infection are usually derived from B lymphocytes. Although a broad spectrum of T-cell-derived malignancies has been described, no case of monoclonal T large granular lymphocyte leukaemia has been reported to date. We report a case of clonal T-LGL (CD3⁺, CD4⁻, CD8⁺, CD56⁻, CD57⁺) in an HIV-infected, HTLV1/2-negative individual. Large granular lymphocytes are thought to represent activated cytotoxic T lymphocytes. HIV infection, as previously reported for HTLV1/2, may represent a pathway of antigen activation and lead to clonal expansion of T large granular lymphocytes.     

Immunophenotype of c-kit cells in normal human bone marrow: implications for the detection of minimal residual disease in AML

Summary. In the present study, seven normal human bone marrow samples from healthy volunteers have been analysed in order to investigate the immunophenotypic characteristics of the normal CD117⁺ cells and their utility for the detection of minimal residual disease in 71 acute myeloid leukaemia patients.
Our results show that most of normal BM CD117⁺ cells coexpress the HLADR and the myeloid associated CD33 antigen. In addition, almost half of CD117⁺ cells are CD34⁺, these cells displaying a different FSC/SSC distribution when compared to the CD117⁺/CD34⁻ cells. No CD117⁺/CD15⁺ and CD117⁺/CD10⁺ cells were detected and very few CD117⁺ cells (<1 × 10⁻³) expressing the HLADR⁻/CD34⁻, CD33⁺/HLADR⁻ and CD34⁺/HLADR⁻ phenotypes were found to be present in normal BM. In contrast, from the 71 AML patients analysed, 34 had CD117⁺/CD15⁺ blast cells and eight had the CD117⁺ phenotypes detected at low frequencies (<1 × 10⁻³) in normal BM.
In summary, the present study shows that the use of the CD117 antigen in different monoclonal antibodies combinations may be of great help for the detection of minimal residual disease in a high proportion of AML cases, especially in those patients displaying the CD117⁺/CD15⁺ phenotype, because cells coexpressing both antigens in normal BM, if present, are at very low frequencies.     

A Family Study of Coeliac Disease

Summary: Studies by Thompson¹, Carter et al. ², MacDonald et al. ³ and McCrae⁴ have all shown a high familial frequency of coeliac disease (CD). The diagnostic criteria differed in each study; in Thompson's patients the diagnosis was based on clinical or historical data alone; in Carter's study response to a gluten-free diet was required, while in MacDonald's and McCrae's studies the diagnostic criteria included the demonstration of the lesion of CD by small bowel biopsy. In MacDonald's study some asymptomatic cases were discovered and a few of these had normal fat balance results. No hereditary pattern emerged from these studies though MacDonald postulated a dominant gene with variable penetrance while McCrae suggested that susceptibility to CD is inherited multifactorially and that environmental factors other than dietary gluten are of aetiological importance. Hoffman et al. ⁵, on the other hand, have described discordance for CD in identical twins.     

Epitope mapping of human factor IX inhibitor antibodies

Summary. We have determined the location of epitopes on the factor IX for three haemophilia B inhibitor antibodies (HB-1, HB-3, HB-7) and a monoclonal anti-factor IX inhibitory antibody (designated 65–10). The main binding region of HB-1, HB-3 and HB-7 was ¹⁵⁵YVNSTEAETI¹⁶⁴ (residues 155–164), ¹⁶⁷NITQSTQSFN¹⁷⁶ and ¹⁵⁶VNSTEAETI¹⁶⁴, respectively. The binding region of 65–10 was ¹⁶⁸ITQSTQSFNDFTRVV¹⁸², which included the cleavage site (¹⁸⁰R-V¹⁸¹) for activation by factor XIa. By neutralization experiments using two peptides, ¹⁵⁶VNSTEAETI¹⁶⁴ and ¹⁶⁷NITQSTQSFN¹⁷⁶, the degree of neutralization of anti-factor IX IgG purified by protein A was determined. Neutralization of three antibodies, HB-1, HB-3 and HB-7, in the presence of 10m m of the peptides ¹⁵⁶VNSTEAETI¹⁶⁴ was 30.1%, 0% and 10.8%, respectively, and in the presence of 4 m m of ¹⁶⁷NITQSTQSFN¹⁷⁶ it was 0%, 13.5% and 17.3%, respectively. On the other hand, when plasmas of patients instead of purified IgG were used for neutralization, 10 m m of ¹⁵⁶VNSTEAETI¹⁶⁴ and 4 m m of ¹⁶⁷NITQSTQSFN¹⁷⁶ failed to neutralize the inhibitor in the plasmas.     

Eosinophilia associated with proliferation of CD3+4−8−αβ+ T cells with chromosome 16 anomalies

We describe a patient with eosinophilia and an abnormal CD3⁺4⁻8⁻αβ⁺ T-cell population. Chromosomal analysis of sorted CD3⁺4⁻8⁻ cells revealed abnormal karyotypes on chromosome 16. In the presence of IL-2 the production of IL-5 from CD3⁺4⁻8⁻ cells was higher than that from CD3⁺4⁺/8⁺ cells. Eosinophil survival-enhancing activity in the patient serum was inhibited by a combination of anti-IL-5 and anti-GM-CSF monoclonal antibodies. These data suggest that increased production of IL-5 and GM-CSF from the abnormal CD3⁺4⁻8⁻ cells might cause eosinophilia.     

Major role for CD8+ T cells in the protection against Toxoplasma gondii following dendritic cell vaccination

Toxoplasma gondii is the causative agent of toxoplasmosis, a worldwide zoonosis for which an effective vaccine is needed. Vaccination with pulsed dendritic cells is very efficient but their use in a vaccination protocol is unconceivable. Nevertheless, unravelling the induced effector mechanisms is crucial to design new vaccine strategies. We vaccinated CBA/J mice with parasite extract-pulsed dendritic cells, challenged them with T. gondii cysts and carried out in vivo depletion of CD4⁺ or CD8⁺ T lymphocytes to study the subsequent cellular immune response and protective mechanisms. CD4⁺ lymphocytes were poorly implicated either in spleen and mesenteric lymph node (MLN) cytokine secretion or in mice protection. By contrast, the increasing number of intracerebral cysts and depletion of CD8⁺ cells were strongly correlated, revealing a prominent role for CD8⁺ lymphocytes in the protection of mice. Splenic CD8⁺ lymphocytes induce a strong Th1 response controlled by a Th2 response whereas CD8⁺ cells from MLNs inhibit both Th1 and Th2 responses. CD8⁺ cells are the main effectors following dendritic cell vaccination and Toxoplasma infection while CD4⁺ T cells only play a minor role. This contrasts with T. gondii infection which elicits the generation of CD4⁺ and CD8⁺ T cells that provide protective immunity.     

Germinal centre and marginal zone B cells expand quickly in a second Plasmodium chabaudi malaria infection producing mature plasma cells

Antibodies and B cells are critical in the protective immune response to the blood stage of the malaria parasite, Plasmodium chabaudi. However, little is known about the development of memory B cells and their differentiation into plasma cells during infection or after re-infection. Here we have shown that B cells with phenotypic characteristics of memory cells (CD19 ⁺ IgD⁻ CD38 ⁺ , IgG1 ⁺ ) are generated in a primary Plasmodium chabaudi chabaudi infection of mice. In addition, we observed that germinal centre cells (CD19⁺, GL7⁺, MHCII^hi) and Marginal Zone B cells (CD19⁺CD23⁻IgD⁻) show faster expansion on re-infection than in the primary, though other subsets do not. Interestingly, though both IgM⁻ and IgM⁺ memory cells are produced, IgM⁺ memory cells do not expand on second infection. The second infection quickly produced mature bone marrow plasma cells (intracellular Ig^hi, CD138^hi, CD9⁺, B220⁻), compared to primary infection; which generates a very large population of immature splenic plasma cells (B220+). This analysis suggests that a memory B cell population is generated after a single infection of malaria, which on re-infection responds quickly producing germinal centres and generating long-lived plasma cells making the second encounter with parasite more efficient.     

Expression of CD8β and alteration of cell surface phenotype in adult T-cell leukaemia cells

Typical adult T-cell leukaemia (ATL) cells have a CD4⁺CD8⁻ cell surface phenotype, but atypical phenotypes such as CD4⁺CD8⁺ and CD4⁻CD8⁺ have also been reported. The CD8 molecule is composed of α and β chains and commonly used monoclonal antibodies against CD8 molecule detect only CD8α. Since it has been reported that CD8α can be induced in mature CD4⁺ T cells by cell activation, but not CD8β, we studied whether ATL cells which express CD8α may also express CD8β. We found some cases of CD8α⁺ ATL were also positive for CD8β. Furthermore, we experienced a case whose ATL cell surface phenotype changed from CD4⁺CD8α⁺CD8β⁺ to CD4⁻CD8α⁺CD8β⁺ and finally to CD4⁺CD8α⁻CD8β⁻. Southern blot analysis revealed that the monoclonal integration of human T lymphotropic virus type I (HTLV-I) was identical throughout the course of the study, indicating that a single clone had demonstrated the alterations. These data suggest that peripheral CD4⁺CD8⁺ ATL cells can express not only CD8α, but also CD8β and that a single ATL cell clone has the potential to change its surface phenotype in vivo as well as in vitro .     

Monoclonal proliferation of CD4+ large granular lymphocytes with cytolytic activity

Summary. A rare case of monoclonal proliferation of CD3⁺4⁺8∼ T-cell receptor-αβ⁺ large granular lymphocytes (LGL) is presented. CD4⁺ LGL in the present case showed spontaneous cytotoxicity against herpes simplex virus-infected cells and antibody- and lectin-dependent cytotoxicity. Perforin, which is one of the important cytolytic mediators of cytotoxic T cells (CTL) and natural killer cells, was abundantly expressed in CD4⁺ LGL of this case. The present case suggests that perforin-positive CD4⁺ CTL, which have recently been shown in the in vitro studies, certainly exist in vivo.     

Monitoring of CD95 and CD38 expression in peripheral blood T lymphocytes during active human cytomegalovirus infection after orthotopic liver transplantation

Aim:  The aim of the present study was to quantitatively monitor the response of CD95 molecules expressed on CD3⁺ T cells (CD95⁺CD3⁺ cells) and CD38 molecules expressed on CD8⁺ T cells (CD38⁺CD8⁺ cells) to ganciclovir treatment after orthotopic liver transplant (OLT) in recipients with active human cytomegalovirus (HCMV) infection.
Methods:  Blood samples were collected from 20 liver transplanted recipients with active HCMV infection and 24 recipients without HCMV infection. CD95⁺CD3⁺ cells and CD38⁺CD8⁺ cells were quantitatively detected with QuantiBRITE bead methods by dual-color flow cytometry analysis during the post-transplantation period.
Results:  CD95⁺CD3⁺ cells and CD38⁺CD8⁺ cells were not significantly different among different ages of healthy adults ( P  > 0.05). CD95⁺CD3⁺ cells and CD38⁺CD8⁺ cells were drastically increased in the active HCMV infection group compared with that in the stable group or in the healthy group ( P  < 0.001), and then they were gradually decreased within the next several weeks after ganciclovir treatment when compared with active HCMV infection recipients ( P  < 0.001).
Conclusions:  The present study showed that CD38⁺CD8⁺ T cells can be an appropriate immunological marker for early detection and antiviral therapeutic monitoring of HCMV infection. The evaluation of CD95 molecule levels may be used routinely in clinical practice to assess the level of immunosuppression.     

Epidemiology of Crohn's Disease in Southern Israel

Objectives: Crohn's disease in Israel was described in the past as being of low incidence, more common in Europe-America-born Jews than other Jews, and of unebaracteristically low morbidity. However, recent experience bas suggested that these premises are no longer correct. Methods: the records of all hospital and outpatient cases of Crobn's disease in soutbern Israel for the period 1968–1992 were reviewed. Private family practitioners and specialists were contacted to assure complete case ascertainment. Results: the prevalence rate of Crohn's disease among Jews on December 31, 1992, was 50.6/10⁵ (Asia-Africa-born Jews 55.0/10⁵, Europe-America-born Jews 58.7/10⁵), and the rate was 8.2/10⁵ among Bedouin Arabs. the annual incidence rate (1987–1992) was calculated as 4.2/10^%r in Jews (Asia-Africa-born 4.6/10⁵/yr, Europe-America-born 3.9/10⁵/yr). the age of presentation declined progressively over the study period, was lower in Israel-born patients than immigrants, and was lower in ileocolonic versus other sites of disease. Conclusions: The data show that Crobn's disease bas become more common in Jews in Israel, losing ethnic differences of frequency, and that it occurs at a younger age than before. In Arabs, the disease is more rare.     

Comparison of Haematological Features of the β0 and β+ Thalassaemia Traits in Jamaican Negroes

Haematological characteristics have been compared in 29 subjects with heterozygous β⁰ thalassaemia and in 33 subjects with heterozygous β⁺ thalassaemia, identified by the type of sickle cell-β thalassaemia among close relatives, in a Jamaican Negro population. Total haemoglobin, MCV and MCH were significantly lower in the β⁰ type but the level of Hb A₂ was not significantly different. Individual values for MCV, MCH and Hb A₂ in the β⁺ type occasionally overlapped those in the normal population casting doubt on the adequacy of these criteria in identifying all cases of heterozygous β⁺ thalassaemia. The haematological differences are those which would be expected on theoretical grounds. The inability to confidently differentiate the two types of heterozygous β thalassaemia has implications for genetic counselling. The inability to distinguish heterozygous β⁺ thalassaemia from normals on any single haematological index suggests that surveys depending on estimations of Hb A₂ or on MCV alone may have underestimated the prevalence of the β⁺ thalassaemia gene.     

Haemopoietic progenitor cell differentiation: flow cytometric assessment in bone marrow and thymus

Summary. We have recently shown that expression of any of the lineage-associated molecules CD2, CD7, CD10, CD19 or CD33 does not ensure lineage-commitment of CD34⁺ progenitor cells. Further, normal progenitor cells and leukaemic blast cells have been shown to coexpress molecules associated with more than one haemopoietic lineage. Five-dimensional flow cytometric analysis of normal bone marrow cells was exploited to investigate the hypothesis of a developmental stage in haemopoiesis comprising CD34⁺ cells coexpressing CD2, CD5, CD7, CD10, CD19 and CD33 or any combination of these molecules. We report on a subpopulation of CD34⁺ bone marrow cells constituting < 5% of the CD34⁺ cells and characterized by extensive coexpression of several molecules associated with the B lymphoid, T lymphoid and myeloid lineages. There is every probability that some cells display the CD34+ CD2⁺ CD5⁺ CD7⁺ CD10⁺ CD19+ CD33+ phenotype. Studies on postnatal thymocytes suggest that this may be the phenotype or one of a few phenotypes of a candidate thymus-seeding progenitor cell population. Finally, our findings that CD34⁺ as well as CD34⁺ CD5⁺ thymocytes can be driven into non-T-lymphoid differentiation by cytokines, support the notion that the thymus is seeded by uncommitted progenitors.     

CD3-induced T-cell activation in the bone marrow of myeloma patients: major role of CD4+ cells

Summary. A large expansion of activated T cells (CD3⁺CD25⁺) with the potential to act as anti-tumour effector cells is inducible in multiple myeloma (MM) patients by culturing bone marrow mononuclear cells (BMMCs) with the anti-CD3 monoclonal antibody (mAb) OKT3. The aim of this study was to provide a greater characterization of CD3-activated T cells. On day 6, most T cells coexpressed the CD1 la, CD18, CD54, CD45R0 antigens and consisted of activated (CD25⁺) CD4⁺ and CD8⁺ cells in nearly equal proportions. Kinetics studies showed that CD4⁺CD25⁺ cells proliferated more rapidly and peaked earlier than CD8⁺CD25⁺ cells. When experiments were performed with purified subpopulations by removing CD4⁺ cells (resulting in CD8⁺ BMMCs) or by removing CD8⁺ cells (resulting in CD4⁺ BMMCs), T-cell activation and autologous plasma cell decrease were observed in CD4⁺ BMMCs only. Transwell cultures showed that CD4 help was necessary to make CD8⁺ BMMCs susceptible to CD3 stimulation. Relevant amounts of IL-2 were found in the supernatants of CD4⁺ BMMCs cultures, whereas no secretion of IL-4 was detected, indicating a Thl-like profile of CD3-activated CD4⁺ cells.
These data indicate that CD4⁺ cells proliferate earlier and provide optimal help to induce the subsequent expansion of CD8+ cells after CD3 stimulation of MM BMMCs. Adequate stimulation of CD4⁺ cells is therefore essential in any strategy aiming to recover T-cell-mediated immunity in MM.