HIV viraemic patients downregulate CD94/NKG2A inhibitory receptors on NK as well as CD8 T cells in comparison with aviraemic counterparts

Background: The CD94/NKG2 heterodimer is a C-type lectin receptor formed by the association of CD94 and one of the NKG2 molecules (namely NKG2A, -B, -C or –E). The interaction of CD94/NKG2A with non classical HLA-E molecules delivers inhibitory signals. CD94/NKG2A is normally expressed on most NK cells whereas less than 5% of peripheral resting CD8+ T cells are positive. Although several reports have clearly shown an upregulation of CD94 on CD8 T cells in HIV infection, the simultaneous expression of both subunits of the inhibitory receptor on NK and T cells and its relation with viral load is largely unknown. Methods: PBLs from 30 HIV-infected patients (16 viraemic and 14 aviraemic under HAART) and 18 healthy volunteers were analysed by flow cytometry after staining with the following monoclonal antibodies (Percp-conjugated anti-CD8, FITC-conjugated anti-CD3, APC-conjugated anti-CD94, PE-conjugated anti-NKG2A). Results: The proportion of CD8 T cells expressing the CD94/NKG2A inhibitory receptor was not significantly increased in HIV-infected patients (5.68 ± 3.72%) in comparison with non-infected controls (4.90±2.84%). Interestingly, patients with viral load < 50 copies/ml had a higher proportion of CD8 T cells expressing the inhibitory receptor (7.15 ± 3.63%) than patients with HIV viraemia (4.40 ± 3.40%), p= 0.041. The same pattern was observed for NK cells and was even more pronounced. In aviraemic individuals, 61.75 ± 20.39% of NK cells expressed the inhibitory receptor vs 42.88 ± 26.38% in viraemic patients. The proportion of CD94/NKG2A positive cells was correlated between NK and CD8 T cell subsets (p=0.0351) but there was no correlation with absolute or relative CD4 counts. Conclusions: Our results suggest that chronic stimulation with HIV antigens in viraemic patients could lead to decreased rather than increased expression of inhibitory receptors on NK and CD8 T cells. This could contribute to the abnormal activation of the immune system associated with advanced HIV disease

Low T-cell chimerism is not followed by graft rejection after nonmyeloablative stem cell transplantation (NMSCT) with CD34-selected PBSC.

peer reviewedWe investigate the feasibility of CD34-selected peripheral blood stem cell (PBSC) transplantation followed by pre-emptive CD8-depleted donor lymphocyte infusions (DLI) after a minimal conditioning regimen. Six patients with advanced hematological malignancies ineligible for a conventional myeloablative transplant (n=5) or metastatic renal cell carcinoma (n=1), and with an HLA-identical (n=4) or alternative (n=2) donor were included. The nonmyeloablative conditioning regimen consisted in 2 Gy TBI alone (n=4), 2 Gy TBI and fludarabine (RCC patient, n=1) or cyclophosphamide and fludarabine (patient who had previously received 12 Gy TBI, n=1). Post transplant immunosuppression was carried out with cyclosporin (CyA) and mycophenolate mofetil (MMF). Initial engraftment was achieved in all patients. One out of six patients (17%) experienced grade > or =2 acute GVHD only after abrupt cyclosporin discontinuation and alpha interferon therapy for life-threatening tumor progression. T-cell chimerism was 23% (19-30) on day 28, 32% (10-35) on day 100, 78% (49-95) on day 180 and 99.5% (99-100) on day 365. Three out of four patients who had measurable disease before the transplant experienced a complete response. We conclude that CD34-selected NMSCT followed by CD8-depleted DLI is feasible and preserves engraftment and apparently also the graft-versus-leukemia (GVL) effect. Further studies are needed to confirm this encouraging preliminary report

Downregulation of CD94/NKG2A inhibitory receptors on CD8+ T cells in HIV infection is more pronounced in subjects with detected viral load than in their aviraemic counterparts

The CD94/NKG2A heterodimer is a natural killer receptor (NKR), which inhibits cell-mediated cytotoxicity upon interaction with MHC class I gene products. It is expressed by NK cells and by a small fraction of activated CD8+ T lymphocytes. Abnormal upregulation of the CD94/NKG2A inhibitory NKR on cytotoxic T cells (CTLs) could be responsible for a failure of immunosurveillance in cancer or HIV infection. In this study, CD94/NKG2A receptor expression on CD8+ T lymphocytes and NK cells was assessed in 46 HIV-1-infected patients (24 viraemic, 22 aviraemic) and 10 healthy volunteers. The percentage of CD8+ T lymphocytes expressing the CD94/NKG2A inhibitory heterodimer was very significantly decreased in HIV-1-infected patients in comparison with non-infected controls. Within the HIV infected patients, the proportion of CD8+ T lymphocytes and NK cells expressing CD94/NKG2A was higher in subjects with undetectable viral loads in comparison with their viraemic counterparts. No significant difference was detected in the proportion of CD8+ T lymphocytes expressing the activatory CD94/NKG2C heterodimer between the HIV-1 infected patients and the healthy donors, nor between the vireamic and avireamic HIV-1 infected patients. In conclusion, chronic stimulation with HIV antigens in viraemic patients leads to a decreased rather than increased CD94/NKG2A expression on CD8+ T lymphocytes and NK cells

2002; 01

DLI is feasible with rapid engraftment and minimizes the risk of severe GVHD. Large prospective trials are required to prove that it preserves the GVL effect fully. ©2002, Ferrata Storti Foundation Key words: allogeneic transplantation, GVHD, GVL, CD34 selection, donor lymphocyte infusions. plication of DLI is GVHD, which is often associated with response to DLI. © F e r r a t a S t o r t i F o u n d a t i o n Design and Methods Patients and donors Twenty-four patients with hematologic malignancies, 18 males and 6 females, aged 14 to 56 years (median 46 years) were included. Their clinical characteristics are summarized in © F e r r a t a S t o r t i F o u n d a t i o n obtained from patients and donors and our institution&apos;s Ethical Committee approved the protocol. Clinical management The conditioning regimen depended on the clinical diagnosis. The following regimens were used: 1) Ara-C (12 g/m 2 ), cyclophosphamide (120 mg/m 2 ) and single dose total body irradiation-TBI (8 Gy) (AML and MDS patients); 2) Ara-C (18 g/m 2 ), melphalan (140 mg/m 2 ) and fractionated TBI (12 Gy) (ALL patients); 3) busulfan (16 mg/kg) and cyclophosphamide (120 mg/kg) (CML and 2 nd transplant patients); 4) cyclophosphamide (120 mg/kg) and fractionated TBI (12 Gy) (NHL and CLL patients). All patients were treated with 5 µg/kg/d lenograstim (Granocyte®) from day +1 until the granulocyte count was &gt; 10 9 /L for three consecutive days or &gt; 10 10 /L for one day. Six early patients (4 not HLA-identical to their donor and 2 AML in CR1) received a short course of methotrexate in addition to cyclosporine (CyA). Because of the low incidence of acute GVHD observed in the first 9 patients, GVHD prophylaxis was carried out with CyA alone for patients 10 to 24. The diagnosis and grading of acute and chronic GVHD was established as previously reported. Stem cell mobilization, collection and selection Donors received human granulocyte colonystimulating factor (G-CSF) (Granocyte®, kindly provided by Rhône-Poulenc-Rorer, Brussels, Belgium) at a dose of 10-15 µg/kg from day -5 through day -1 before transplant. Collection of PBSC was carried out on days -1 and 0, using a continuous flow blood cell separator (CS3000+, Baxter-Fenwall Laboratories, Deerfield, IL, or Cobe Spectra, Lakewood, CO, USA). The volume of blood processed was 12-16 liters. The PBSC from the first day of harvest were stored overnight in the patient&apos;s own plasma. Immediately after the second harvest, PBSC from the first and the second days of harvest were pooled. CD34 + cell selection was carried out using the Isolex 300i® magnetic cell separator (Nexell International, Wemmel, Belgium), according to the manufacturer&apos;s recommendations. Donor lymphocyte infusions Around day 60 post-transplantation, donors underwent 12-16 liter leukophereses on 2 consecutive days to collect lymphocytes. The collection from the first day of harvest was stored overnight in the patient&apos;s own plasma and pooled with the second harvest before processing by CD8 + selection using the Nexell Isolex 300i®. The CD8-negative fraction was recovered and divided into 3 aliquots containing 2×10 6 , 1×10 7 and 5×10 7 CD3 + cells/kg recipient for patients from #1 to #13. The 2 nd and 3 rd aliquots were cryopreserved in 10% DMSO in a controlled-rate freezer. After an interim analysis showed little acute or chronic GVHD associated with DLI, the schedule of DLI was changed to 2 aliquots of 1×10 7 and 5×10 7 CD3 + cells/kg recipient for patients 14 to 21. Aliquot 1 was injected fresh immediately after the CD8 depletion procedure (around day 60). Around day 100 (and 140 for patients 1 to 13), aliquot 2 (and 3) were thawed and infused into the patient. CD8-depleted DLI were not to be infused in case of an antecedent grade III or IV acute GVHD, or an antecedent extensive chronic GVHD, or active GVHD at the time of the scheduled infusion. Laboratory analyses Aliquots of the pooled PBSC as well as the CD34 + selected fraction were incubated with phycoerythrin-conjugated anti-CD34 monoclonal antibody (HPCA2; Becton-Dickinson, Palo-Alto, CA, USA) for 20 minutes at 20°C, washed and fixed. A total of 1×10 5 cells was analyzed using a FACS-scan analyzer (Becton-Dickinson). The percentage of CD34 + cells was defined with dot plot analysis using the whole nucleated cell population. The percentage of positive cells in the isotype control was subtracted from the CD34 + percentage to give the final percentage of CD34 + cells. Data acquisition was performed with the Cellquest software (BectonDickinson). Donor lymphocytes (before and after CD8 depletion) were similarly examined using double labeling with FITC-and PE-conjugated antibodies after treatment with a lysing solution. Complete blood counts were determined using a Technicon H2 cell counter (Bayer Diagnostics, Diegem, Belgium). Percentages of reticulocytes were obtained by an automated cytofluorometric method using the thiazole orange analog DEQTC. Statistical analyses Student&apos;s t-tests were used to compare cell subsets before and after CD8 depletion. The probability of GVHD, relapse, and survival as well as the speed of engraftment were studied by life-table analyses and Wilcoxon rank tests were used for comparisons between groups. Statistical analyses were carried out with Graphpad Prism (Graphpad Software, San Diego, CA, USA). Results Collection of PBSC, CD34 selection and engraftment kinetics PBSC were collected by leukophereses on two consecutive days, except in one 70-year old donor (patient #6) who had to undergo 4 consecutive leukophereses (and two CD34 selection procedures) because of poor yields. Most donors experienced bone pain and/or cephalalgia that were easily controlled with paracetamol, but no other complication was noted. A median of 10.15 (5.59 to 21.02) ×10 6 CD34 + cells/kg and 375 (127 to 656)×10 6 CD3 + cells/kg were collected CD8-depleted DLI after CD34-selected allo-PBSCT © F e r r a t a S t o r t i F o u n d a t i o n Collection of donor lymphocytes, CD8 depletion and CD8-depleted DLI Donor lymphocytes were collected on day 60 from all but 5 donors whose recipients died before or experienced serious complications Clinical data (Figure 1 and Table 4) Five patients died before receiving DLI. Patients #6 (second transplant) and #4 developed many early serious complications and died on day 81 of influenza pneumonia and on day 99 of a polymicrobial infection, respectively. Patient #19 developed severe renal and respiratory failure and died of infection at day 195. Patient #16 died of CLL and veno-occlusive disease of the liver on day 44 and patient #17 died of leukemia on day 38. The other 19 patients were in CR on day 60 and received the first DLI. Two ALL patients (patients #7 and 10) relapsed between days 60 and 100 and received the pooled 2 nd and 3 rd aliquots. Patient #7 achieved a CR with chemotherapy and DLI but relapsed later and died on day 313. Patient #10 died of leukemia on day 100. In addition, patient #12 died suddenly at home of unknown reason while enjoying continuous CR and presenting no complication other than depression. The other 16 received the scheduled 2 nd (and the 3 rd for patients #1, 2, 3, 5, 8, 9, 10, 13) DLI on days 100 (and 140). Fourteen of the 16 patients remain in CR 126 to 1344 (median 400) days post-transplantation. Patient #15 developed a central nervous system relapse on day 135, was reinduced into remission with chemotherapy and radiotherapy but relapsed again and died on day 309. Patient #3 experienced a biopsyproven massive relapse in the thymus on day 315 that completely regressed after cyclosporine discontinuation, but later relapsed again and she died on day 950. Finally, 3 patients died in CR after day 100: patient #13 died of pulmonary aspergillosis on day 351, patient #18 died suddenly at home of unknown reason on day 288 and patient #21 of acute grade IV GVHD on day 150. The remaining 11 patients were alive and disease-free 126 to 1344 (median 550) days after transplantation. Bone marrow chimerism Evaluation of bone marrow chimerism was performed in 6/7 patients with a donor of the opposite sex (n=7) by FISH with X and Y probes ( Acute and chronic GVHD (Figure 3) Acute GVHD occurred in 15 patients. It was of grade I in 9 patients, of grade II in 5 patients (3 with 1 HLA mismatch) and of grade IV in 1 patient (HLA mismatch). Thus, grade II-IV acute GVHD occurred more frequently in HLA-mismatched (4/8 or 50%) than HLA-matched (2/16 or 13%) transplants (p&lt;0.05). Before DLI, the 60-day actuarial incidence of grade II-IV GVHD was 17% (4 patients) but 2 additional patients developed grade II and IV GVHD after DLI to produce a 150-day (after DLI) incidence of 28%. For HLA-identical sibling transplants, the 60-and 150-day actuarial incidences of grade II-IV acute GVHD were 0 and 13%, respectively ( CMV reactivation Eleven of the 24 patients experienced CMV reactivation (PCR positivity) before day 60 that was successfully reversed by ganciclovir treatment and none of them presented a clinical CMV infection. Relapse and survival (Figure 4) There was no relapse in the 12 standard risk patients but 6 of the high-risk patients relapsed (p&lt;0.005) ( Discussion In agreement with previous studies, 17,19,21 CD34-selection resulted in our study in a 3.3 log elimination of T-cells while preserving hematologic reconstitution. Engraftment of neutrophils and platelets was prompt and significantly faster in patients who did not receive methotrexate. However, within the range studied, the number of © F e r r a t a S t o r t i F o u n d a t i o n CD34 + cells did not influence the speed of engraftment significantly. Prompt engraftment occurred with CD34 + cell doses as low as 1.46×10 6 /kg. Therefore, CD34 -selection preserved the engraftment capability of allogeneic PBSC. As previously suggested by others, 17,22 our results evidenced that CD34 selection reduces the risk of acute GVHD. The actuarial 60-day (before DLI) probability of grade II-IV acute GVHD was 17% in our study. This rate compares very favorably with results from studies of HLA-identical siblings receiving unmanipulated PBSC or BM. Moreover, our results also compared favorably with those of previous studies of pre-emptive DLI after T-cell-depleted BMT or PBSC transplantation. A few other studies have investigated the feasibility of adding T-cells back, also to heterogeneous groups of patients, a few weeks to a few months after T-cell-depleted (TCD) transplantation. Barrett gave either 2×10 6 /kg on day 30 and 5×10 7 /kg on day 45 or 1×10 7 /kg on day 30 to HLA-identical siblings after TCD BMT. Contributions and Acknowledgments YB designed the study and wrote the paper. FB analyzed the data and wrote the paper. JS © F e r r a t a S t o r t i F o u n d a t i o n PEER REVIEW OUTCOMES What is already known on this topic Transplantation of CD34 + selected cells from peripheral blood of allogeneic donors associates with a lower risk of acute and chronic graft-versus-host disease than unmanipulated transplants. Relapse risk may be increased in this setting, as a consequence of T-cell depletion, which leads to a decreased graft-versusleukemia effect. What this study adds CD8-depleted lymphocyte infusions starting on day 60 may be safely administered without triggering acute or chronic graft-versus-host disease. The relapse rate after this approach was similar to that observed in a control group of non-T-cell depleted transplants. Manuscript processing Potential implications for clinical practice This report demonstrates the feasibility of allogeneic CD34 + selected stem cell transplantation followed by the infusion of engineered cell-depleted fractions. Jordi Sierra Gil, Deputy Editor © F e r r a t a S t o r t i F o u n d a t i o

Translocation (2;3)(p21;q26) as the sole anomaly in a case of primary myelofibrosis.

peer reviewedTranslocation t(2p;3q) is a rare but recurrent finding in myeloid disorders. We present the first case of primary myelofibrosis with t(2;3)(p21;q26) as the sole chromosomal anomaly. The comparison with the 11 other previously published myeloid-associated t(2p;3q) cases confirms that this nonrandom translocation involves a pluripotent stem cell and is associated with a poor prognosis

Clinical case of the month. Cholestatic hepatitis after administration of piperacillin

peer reviewedWe describe a patient suffering from infection of the upper respiratory tract, who was treated with a dose of 4 x 4 g of piperacillin over 10 days. Two days after the end of the treatment, she developed jaundice and had elevated alkaline phosphatase, gammaglutamyltransferase and transaminases. After exclusion of viral hepatitis, a vascular problem, and gall stone disease, the possibility of piperacillin-induced hepatitis was discussed. Lymphocyte transformation test for piperacillin was positive, suggesting an immunological mechanism for the observed hepatopathy. The patient was discharged a few days after in good clinical condition and with reduced liver values. Cholestasis gradually decreased but was detectable for several weeks. The patient had a full clinical and biochemical recovery after 2 months. We conclude that short-term therapy with piperacillin can lead to the same type of hepatopathy as described for amoxycillin/clavulanic acid or antistaphylococcal penicillins. Positive lymphocyte transformation is compatible with an immunological mechanism

Soluble Interleukin-2 Receptor in Crohn's Disease. Assessment of Disease Activity and Prediction of Relapse

In Crohn's disease, the activity of the disease is difficult to evaluate and the evolution of the disease is difficult to predict. The soluble interleukin-2 receptor serum level has been reported to correlate with clinical activity of the disease and with mucosal immune activation. We compared serum soluble interleukin-2 receptor to classical inflammatory markers and other immune parameters in the assessment of clinical disease activity and prediction of relapse in patients with Crohn's disease. Soluble interleukin-2 receptor serum levels correlated well with the Crohn's disease activity index, and multivariate analysis showed that this correlation was independent of the other inflammatory and immune markers. The correlation was not greater, However, than that between some inflammatory markers, such as ESR, and Crohn's disease activity index. Longitudinal follow-up showed that a high soluble interleukin-2 receptor serum level was highly predictive of relapse. Multivariate analysis showed that the soluble interleukin-2 recepteur serum level was complementary to other inflammatory and clinical markers in the prediction of relapse of disease. We conclude that soluble interleukin-2 receptor is of use in monitoring Crohn's disease, particularly in prediction of relapse

Further Characterization of Cytotoxic T Cells Generated by Short-Term Culture of Human Peripheral Blood Lymphocytes with Interleukin-2 and Anti-Cd3 Mab

In this study we have specifically investigated the participation of T cells in the cytotoxic activity of peripheral blood lymphocytes (PBL) activated by interleukin-2 (IL-2, 50 U/ml) alone or in combination with an anti-CD3 mAb (BMA030, 10 ng/ml, IgG2a). Purified CD3+ T cells, incubated in the presence of the anti-CD3 mAb for 4 days, mediated a cytotoxic activity against HL60 and U937 tumor cell lines. Several findings suggested the involvement of a redirected-cytotoxicity phenomenon, since the lytic process was restricted to target cell lines bearing the high-affinity Fc gamma receptor (Fc gamma RI) and T lymphocytes stimulated by IL-2 alone did not lyse these cell lines. Furthermore, anti-CD3 mAb F(ab')2, anti-CD3 IgG1 (UCHT1), phytohemagglutinin or staphylococcal enterotoxin A did not induce a similar cytotoxic activity in T lymphocytes. The cytotoxic process occurred in the presence of a very low level of anti-CD3 antibodies (in the nanomolar range). The cytotoxic activity of T cells stimulated by IL-2 or by IL-2 + BMA030, against OVCAR-3 cells (MOv18+ ovarian tumor cell line), was also compared in the presence of a bispecific antibody OC/TR, anti-CD3 x MOv18). The stimulation by IL-2 + BMA030 induced approximately a twofold higher cytotoxic activity than IL-2-activated T cells. This could be related to the state of activation of effector cells stimulated by IL-2 + BMA030, since the phenotypic analysis showed an increased proportion of T cells expressing several activation/differentiation markers (CD25, HLA-DR, CD45R0, adhesion molecules). These findings could be applied to the design of therapeutic protocols using anti-CD3 x antitumoral bispecific antibodies

Systemic Immune Response after Rectocolonic Administration of Ovalbumin in Mice

The aim of our study was to determine the effect of a rectocolonic preimmunization with ovalbumin on the systemic immune response induced by a subsequent subcutaneous injection of this antigen in Balb/c mice. One rectocolonic, but not intragastric, administration of 25 mg of ovalbumin induced a detectable increase in serum anti-ovalbumin antibody level. The level reached was however much lower than after subcutaneous injection. Both intragastric and rectocolonic immunization with ovalbumin induced specific systemic cellular tolerance. However, after rectocolonic, but not intragastric, preimmunization there was no systemic humoral tolerance to this antigen. These differences in systemic immune responses after rectocolonic or intragastric administration of ovalbumin could be due to different stimulation of the systemic immune system or to differences between the colonic and small bowel mucosal immune system, which remain to be elucidated