HLA Class II Antibody Activation of Endothelial Cells Promotes Th17 and Disrupts Regulatory T Lymphocyte Expansion.

Kidney transplantation is the most successful treatment option for patients with end-stage renal disease, and chronic antibody-mediated rejection is the principal cause of allograft loss. Predictive factors for chronic rejection include high levels of HLA alloantibodies (particularly HLA class II) and activation of graft endothelial cells (ECs). The mechanistic basis for this association is unresolved. We used an experimental model of HLA-DR antibody stimulation of microvascular ECs to examine the mechanisms underlying the association between HLA class II antibodies, EC activation and allograft damage. Activation of ECs with the F(Ab')2 fragment of HLA-DR antibody led to phosphorylation of Akt, ERK and MEK and increased IL-6 production by ECs cocultured with allogeneic peripheral blood mononuclear cells (PBMCs) in an Akt-dependent manner. We previously showed that HLA-DR-expressing ECs induce polarization of Th17 and FoxP3(bright) regulatory T cell (Treg) subsets. Preactivation of ECs with anti-HLA-DR antibody redirected EC allogenicity toward a proinflammatory response by decreasing amplification of functional Treg and by further increasing IL-6-dependent Th17 expansion. Alloimmunized patient serum containing relevant HLA-DR alloantibodies selectively bound and increased EC secretion of IL-6 in cocultures with PBMCs. These data contribute to understanding of potential mechanisms of antibody-mediated endothelial damage independent of complement activation and FcR-expressing effector cells.

Reconstitution of regulatory T-cell subsets after allogeneic hematopoietic SCT.

Previous studies on regulatory T-cell (Treg) reconstitution after allogeneic hematopoietic SCT (HSCT) have suggested that, within the GVHD process, imbalance between effector T cells and Tregs may be more important than the absolute numbers of circulating Tregs. No study has analyzed naive vs memory Treg reconstitution in a longitudinal cohort with large numbers of patients. The reconstitution of total and subsets of Treg was prospectively analyzed by flow cytometry in 185 consecutive recipients at 3, 6, 12 and 24 months after allogeneic HSCT. The levels of total, naive and memory Tregs increased, mainly within the memory subset, but remained lower than healthy controls up to 2 years after transplantation. Reduced-intensity conditioning and peripheral blood (PBSC) as the source of stem cells were associated with better 3-month reconstitution. In multivariate analysis, PBSC, recipient age ⩽25 and no anti-thymoglobulin in the conditioning regimen were associated with a better Treg reconstitution. Naive Treg long-term reconstitution was mainly influenced by recipient age. Whereas prior acute GVHD impaired Treg reconstitution, Treg subsets (absolute numbers and frequencies relative to CD4(+) T-cell subsets) at 3, 6 and 12 months after HSCT were not associated with the occurrence of a later episode of chronic GVHD.

Outcome of kidney transplantations performed with preformed donor-specific antibodies of unknown etiology.

The detection of preformed donor-specific alloantibodies (DSA) with multiplex-bead arrays has led to the common observation that individuals without a history of pregnancy, transfusion or transplantation can have circulating anti-HLA antibodies of unknown etiology. We retrospectively analyzed the risk of antibody-mediated rejection (AMR) and graft outcome in 41 kidney transplant recipients with DSA of unknown etiology (DSA cause-unk) at the time of transplantation. Twenty-one patients received a posttransplantation desensitization protocol, and 20 received standard immunosuppressive therapy. The mean number of DSA was 1.4 ± 0.8, ranging from 1 to 5. Complement-dependent cytotoxicity crossmatches were negative for all the patients. Flow cytometry crossmatches were positive in 47.6% of cases. The incidence of acute AMR was 14.6% at 1 year, regardless of the immunosuppressive regimen. No patients experienced graft loss following AMR. At month 12, across the entire population of patients with DSA cause-unk, the outcomes were favorable: the measured glomerular filtration rate was 63.8 ± 16.4 mL/min/1.73 m(2), the screening biopsies showed low frequencies of microvascular inflammation and no transplant glomerulopathy, and graft and patient survival were 100%. In conclusion, patients with DSA cause-unk are able to mount AMR but have favorable 1-year outcomes.

Outcomes, infections, and immune reconstitution after double cord blood transplantation in patients with high-risk hematological diseases.

Double unrelated cord blood transplant (dUCBT) has been used to circumvent cell dose limitation of single UCBT; however, few data are available describing outcomes, infectious disease, and immune recovery. We analyzed 35 consecutive dUCBT recipients with high-risk malignant disorders (n=21) and bone marrow failure syndromes (n=14). Median follow-up was 32 months. Conditioning regimen was myeloablative in 14 and reduced intensity in 21 patients. Median infused nucleated cell dose was 4 × 10(7) /kg. Median time to absolute neutrophil count >0.5 × 10(9) /L was 25 days. Cumulative incidence (CI) of acute grade II-IV graft-versus-host disease was 47%. Estimated overall survival at 2 years was 48%. CI of first viral infections at 1 year was 92%. We observed 49 viral infections in 30 patients, 34 bacterial infections in 19 patients, and 16 fungal or parasitic infections in 12 patients. Lymphocyte subset analyses were performed at 3, 6, 9, and >12 months after dUCBT. Decreased T-cell and B-cell counts with expansion of natural killer cells were observed until 9 months post transplantation. Recovery of thymopoiesis measured by T-cell receptor excision circles was impaired until 9 months after dUCBT, when the appearance of new thymic precursors was observed. Delayed immune recovery and high incidence of infectious complications were observed after dUCBT in patients with high-risk hematological diseases.

Dissociation of caspase-mediated events and programmed cell death induced via HLA-DR in follicular lymphoma.

Human leukocyte antigens (HLA) class II antigen-mediated apoptosis has been documented in antigen-presenting cells and B lymphoproliferations. Characteristics of the apoptosis include rapidity and selectivity for mature cells. Follicular lymphomas are particularly refractory to apoptosis. The B-cell lymphoma Ramos shares characteristics of this subgroup and is insensitive to apoptosis via simple HLA-DR engagement. However, oligomerization of HLA-DR antigens induced caspase activation followed by phosphatidylserine externalization, activation of PKC-delta and cleavage of nuclear lamin B. Mitochondrial injury was also detected. However, inhibition of caspase activation simply delayed the apoptotic phenotype but neither protected against cell death nor prevented mitochondrial injury. The data in this report demonstrate that the requirements for the initiating signal (oligomerization versus engagement) as well as the molecular pathways varies between different B lymphoproliferations despite their common expression of HLA-DR. Finally, blockade of caspase activation in parallel with HLA-DR mAb stimulation could provide a potent autovaccination stimulus by leading to necrotic death of B-cell lymphomas.

Prolonged immune deficiency following allogeneic stem cell transplantation: risk factors and complications in adult patients.

To evaluate the long-term immune reconstitution after allogeneic haematopoietic stem cell transplantation (SCT), we prospectively screened standard immune parameters in a series of 105 patients, at a median time of 15 months after SCT. Analysing lymphoid phenotypes, in vitro immune functions and immunoglobulin levels, we found that, more than 1 year post SCT, cellular and humoral immunity was still altered in a significant number of patients. CD4+ T cells were < 200/microl in one third of patients, and the CD4/CD8 ratio was still reversed in 78% of patients. Almost all patients showed positive T-cell responses against mitogens, but antigen-specific proliferation assays identified 20% to 80% of non-responders. B-cell counts were reconstituted in 61% of the patients, but levels of total immunoglobulins were still low in 59%. In multivariate analyses, human leucocyte antigen (HLA) disparity between donor and recipient and chronic graft-versus-host disease were the leading causes affecting immune reconstitution. Interestingly, cytomegalovirus (CMV) infections were strongly associated with normal CD8+ T-cell counts. Studying the impact of impaired immune reconstitution on the rate of infections occurring in the 6 years following screening, we identified three parameters (low B-cell count, inverted CD4/CD8 ratio, and negative response to tetanus toxin) as significant risk factors for developing such late infections.

Influence of bone marrow graft lymphocyte subsets on outcome after HLA-identical sibling transplants.

OBJECTIVE: The aim of this study was to analyze bone marrow lymphocyte subsets and CD34 cell dose and their influence on the outcomes of bone marrow transplantation. MATERIALS AND METHODS: Forty-eight patients (median age 30 years, range 5-54) receiving HLA-identical sibling bone marrow transplantation for hematologic malignancies were analyzed. RESULTS: Median number (range) of nucleated cells and CD34+ cells infused were 2.4 (0.4-6.0) x 10(8)/kg and 3.5 (0.5-13.0) x 10(6)/kg, respectively. Probability of neutrophil recovery was 97%. In a multivariate analysis, time to neutrophil recovery was shortened when a higher number of CD3/CD8 cells was infused (> or =1.0 x 10(7)/kg) (hazard ratio [HR] = 2.13, p = 0.018); when the patient was female or had negative cytomegalovirus serology (HR = 2.03, p = 0.03; HR = 0.41, p = 0.009; respectively). The incidence of grade II to IV acute graft-vs-host disease (GVHD) was 47%. Infusion of >1 x 10(7) CD4 infused/kg increased the risk of acute GVHD (HR = 2.86, p = 0.03). Nineteen of 40 patients at risk experienced chronic GVHD, the risk of which was increased by diagnosis of chronic leukemia (p = 0.03), <2.0 x 10(8) nucleated cells infused/kg (p = 0.05), and a low number of all lymphocyte subsets, except CD19. Estimated 3-year survival rate was 54%. Risk of death was increased in patients receiving <3.5 x 10(6)CD34 infused/kg (HR = 0.37, p = 0.02). Only six patients relapsed. CONCLUSIONS: A high cell dose of CD3/CD8 is associated with faster neutrophil recovery, whereas a high cell dose of CD4+ cells increases the incidence of acute GVHD. A high number of nucleated cells and CD34+ cells infused was associated with decreased risk of chronic GVHD and improved survival, respectively.

Immune recovery under highly active antiretroviral therapy is associated with restoration of lymphocyte proliferation and interferon-gamma production in the presence of Toxoplasma gondii antigens.

The in vitro lymphocyte proliferative response (LPR) and interferon (IFN)-gamma production in the presence of Toxoplasma antigen were evaluated in 97 human immunodeficiency virus (HIV)-1-infected patients with CD4 cell counts of <100 cells/microL (group 1), currently >300 cells/microL but previously <100 cells/microL (group 2), or always >300 cells/microL (group 3) and in 28 non-HIV-infected blood donors (group 4), all seropositive to Toxoplasma. In group 2, 81% of patients had a positive LPR, versus 20% in group 1 (P<10(-3)). IFN-gamma production was greater in group 2 than in group 1 (922 vs. 0 Deltapg/mL; P=10(-4)). Multivariate analysis found a significant association between a positive LPR to Toxoplasma antigen and a CD4 count >300 cells/microL (odds ratio [OR], 16.3; 95% confidence interval [CI], 5.3-50.2) and anti-Toxoplasma IgG titer >150 IU/mL (OR, 5; 95% CI, 1.6-15.2). Immune reconstitution under highly active antiretroviral therapy was associated with a restoration of immune responses against Toxoplasma.

Association of STAT1, STAT3 and STAT5 proteins with the IL-2 receptor involves different subdomains of the IL-2 receptor beta chain.

Upon IL-2 stimulation of T lymphocytes, the IL-2 receptor (IL-2R) becomes phosphorylated on specific tyrosine residues which serve as docking sites for proteins containing SH2 or phosphotyrosine binding domains. To study the interaction of the IL-2Rbeta chain with Shc and STAT proteins, subdomains of the IL-2Rbeta chain were expressed as tyrosine-phosphorylated glutathione S-transferase fusion proteins and used to pull-down interacting proteins from Kit 225 cell lysates. These experiments provide direct biochemical evidence that binding to the IL-2R of the adaptor protein Shc requires phosphorylation of Tyr-338 in the IL-2Rbeta acidic subdomain. In addition, we report that STAT proteins that are activated by IL-2, i.e. STAT1, STAT3 and STAT5, indeed associate with the IL-2Rbeta chain. Both the A and B isoforms of STAT5 were found to associate with Tyr-510 of the IL-2Rbeta C-terminal region, depending on its phosphorylation. In contrast, STAT1 and STAT3 associated with the IL-2Rbeta chain through its acidic subdomain. These results indicate that the interaction between IL-2Rbeta and STAT1 or 3 does not require either phosphorylation of the receptor or even the presence of tyrosine residues of IL-2Rbeta. Thus, the IL-2R recruits STAT proteins through different modes of interaction.

Biochemical analysis of interleukin-2 receptor beta chain phosphorylation by p56(lck).

Tyrosine phosphorylation of multiple proteins, including the receptor itself, is an initial event in IL-2 signaling and leads to recruitment of SH2 or PTB domain-containing proteins to the receptor. In this study, we have used subdomains of the IL-2 receptor beta chain (IL-2Rbeta) expressed in Escherichia coli as GST fusion proteins to identify the tyrosine residues that could be phosphorylated by p56(lck), one of the critical tyrosine kinases activated by IL-2. We report that recombinant p56(lck) phosphorylates in vitro tyrosine residues within the IL-2Rbeta chain but not those within the IL-2Rgamma chain. p56(lck) phosphorylates tyrosine residues 355, 358 and 361 but not 338 of the IL-2Rbeta chain acidic subdomain. Interestingly, phosphorylation of Tyr-358 appears to require the presence of either Tyr-355 or Tyr-361. p56(lck) also phosphorylates very efficiently the two tyrosines present in the IL-2Rbeta chain C-terminal region, Tyr-392 and Tyr-510. We also investigated the association of p56(lck) with the IL-2Rbeta chain which was found to depend on a short stretch of the IL-2Rbeta chain acidic subdomain, and to be independent of the presence of its tyrosine residues.

Recent advances in the understanding of interleukin-2 signal transduction.

Interleukin-2 is one of the critical cytokines that control the proliferation and differentiation of cells of the immune system. The present article briefly reviews the current and recently established knowledge on the intracellular signaling events that convert the initial interaction of IL-2 with its receptor into pathways leading to the various biological functions. A first step in IL-2 signaling is the activation of several protein tyrosine kinases that phosphorylate a large array of intracellular substrates including the receptor complex. Phosphorylated tyrosine residues within the receptor then serve as docking sites for multimolecular signaling complexes that initiate three major pathways: the Jak-STAT pathway controlling gene transcription, the Ras-MAPK pathway leading to cell proliferation and gene transcription as well, and the PI3-kinase pathway involved in antiapoptotic signaling and organization of the cytoskeleton. Finally, other recently identified and presumably important tyrosine kinase substrates, whose significance is not yet fully understood, are described.

Protein kinase A phosphorylation of RhoA mediates the morphological and functional effects of cyclic AMP in cytotoxic lymphocytes.

We have observed that stimulation of human natural killer cells with dibutyryl cAMP (Bt2cAMP) reproduced the effects of ADP ribosylation of the GTP binding protein RhoA by Clostridium botulinum C3 transferase: both agents induced similar morphological changes, inhibited cell motility and blocked the cytolytic function. We demonstrate here that cAMP-dependent protein kinase A (PKA) phosphorylates RhoA in its C-terminal region, on serine residue 188. This phosphorylation does not affect the ability of recombinant RhoA to bind guanine nucleotides, nor does it modify its intrinsic GTPase activity. However, treatment of cells with Bt2cAMP results in the translocation of membrane-associated RhoA towards the cytosol. Experiments using purified membrane preparations indicated that Rho-GDP dissociation inhibitor, which can complex phosphorylated RhoA in its GTP-bound state, was the effector of this translocation. Taken together, these data suggest that PKA phosphorylation of RhoA is a central event in mediating the cellular effects of cAMP, and support the existence of an alternative pathway for terminating RhoA signalling whereby GTP-bound RhoA, when phosphorylated, could be separated from its putative effector(s) independently of its GTP/GDP cycling.

Signal transduction of interleukin 2 in human natural killer cells: involvement of the p56lck tyrosine kinase.

Despite numerous reports, the role of the protein tyrosine kinase p56lck in IL-2 signal transduction has remained controversial. We show here, using IL-2-dependent human natural killer cell lines, that p56lck is regulated by IL-2 in two different ways: (1) IL-2 induces a rapid increase of p56lck kinase activity as assessed in vitro; and (2) following IL-2 stimulation, p56lck undergoes phosphorylation on serine residues that is reflected by a modification of its electrophoretic mobility in SDS-PAGE. Furthermore, dose response experiments, and blocking studies performed with anti-IL-2R alpha antibodies, indicated that binding of IL-2 to the IL-2R beta chain was sufficient to produce these modifications of p56lck. In contrast, activation of the CD2 pathway stimulated the kinase activity of p56lck, but did not induce a significant shift in NK cells, as opposed to T lymphocytes. Western blot analyses, and immunoprecipitations of cell lysates from 32P-preloaded NK cells demonstrated that seven major proteins are tyrosine phosphorylated in response to IL-2. These phosphoproteins, with apparent molecular weights of 190, 150, 120, 110, 85, 65 and 56, which may not all be p56lck substrates, undergo phosphorylation and dephosphorylation with different kinetics. Furthermore, pp120 was identified as rasGAP, by Western blot and immunoprecipitation experiments. rasGAP and some of its co-precipitating molecules become phosphorylated in response to IL-2, presumably by p56lck, which would thus provide a link between IL-2R and downstream events critical for NK cell proliferation and function.