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Complement dependent cytotoxicity crossmatch (CDC-XM) has been the original standard crossmatch test, whereas, flow cytometry crossmatch (FCXM) is an enhanced and highly sensitive crossmatch assay performed to detect donor specific anti-HLA antibodies (DSA). We analyzed American Society for Histocompatibility and Immunogenetics (ASHI) proficiency testing data (2011-2020) and examined the number of laboratories performing CDC-XM vs. FCXM, the overall efficiency of laboratories in reporting ≥80% consensus CDC-XM vs. FCXM result, and reasons for non-consensus results in the two assays. Of 600 crossmatches in each crossmatch category, the percentage of laboratories reporting T cell CDC-XMs reduced from 40% in 2011 to 13% in 2020, T cell anti-human globulin (AHG) CDC-XM reduced from 56% in 2011 to 21% in 2020, and B cell CDC-XM reduced from 51% in 2011 to 20% in 2020. The percentage of laboratories performing T cell and B cell FCXM remained at approximately 80% throughout. CDC-XM performed on par with FCXM in providing a consensus negative result using negative DSA serum, but under-performed in comparison to FCXM in providing a consensus positive result using positive DSA serum. In addition, only minority of CDC-XMs was reported positive in presence of complement fixing DSA. This study shows that non-consensus CDC-XM was always in presence of HLA IgG DSA and that laboratories may be struggling to interpret the low sensitive CDC-XM results, where highly sensitive solid phase multi-antigen or single antigen assay shows the presence of HLA IgG DSA in serum.

A T-cell-positive and B-cell-negative flow cytometry crossmatch result remains a conundrum since HLA class I antigens are expressed on both T and B cells. We investigated the frequency, donor HLA specificity of the antibodies, and mechanisms for these crossmatch results.We analyzed 3073 clinical flow cytometry crossmatch tests performed in an American Society of Histocompatibility and Immunogeneticsaccredited histocompatibility laboratory. The sera associated with the T-cell positive and B-cell negative flow cytometry crossmatches were also tested for donor HLA immunoglobulin G antibodies using LABScreen single antigen assays.Among the 3073 test results, 1963 were T-cell negative and B-cell negative, 811 were T-cell negative and B-cell positive, 274 were T-cell positive and B-cell positive, and 25 were T-cell positive and B-cell negative. The LABScreen single antigen assay detected HLA class I immunoglobulin G donor-specific antibodies in 23 of 25 sera associated with a T-cell positive and B-cell negative flow cytometry crossmatch result, and donorspecific antibodies directed at not only HLA-Cw but also at HLA-A or HLA-B were observed. In addition, we identified that the B-cell channel shift threshold used to classify a B-cell flow cytometry crossmatch was a potential contributor to a T-cell-positive and B-cellnegative flow cytometry crossmatch result.Our analysis of 3073 flow cytometry crossmatches, in addition to demonstrating that HLA antibodies directed at the HLA-A, -B, or -Cw locus were associated with a T-cell-positive and B-cell-negative result, identified mechanisms for the surprising T-cell-positive and B-cell-negative flow cytometry crossmatch result.

BackgroundA T cell positive and B cell negative (T+B-) flow cytometry crossmatch (FCXM) result remains a conundrum since HLA-class I antigens are expressed on both T and B cells. We investigated the frequency, HLA specificity of the antibodies and mechanisms for the T+B- FCXM result.MethodsWe analyzed 3073 clinical FCXM tests performed in an American Society of Histocompatibility and Immunogenetics accredited histocompatibility laboratory. The sera associated with the T+B- FCXM were also tested for donor HLA IgG antibodies using LABScreen™ single antigen assays.ResultsAmong the 3073 FCXM tests, 1963 were T-B-, 811 were T-B+, 274 were T+B+, and 25 were T+B-. IgG antibodies directed at donor HLA-A, B, or Cw locus determined antigens (DSA) were identified in all 25 sera and the summed mean fluorescence intensity (MFI) of DSA ranged from 212 to 53,187. Correlational analyses identified a significant association between the summed MFI of class I DSA, and the median channel fluorescence (MCF) of T cells treated with the recipient serum (Spearman rank correlation, rs=0.34, P=0.05) but not with the MCF of B cells (rs=0.23, P=0.24). We identified that differential binding of anti-HLA antibodies to T cells and B cells and the B cell channel shift threshold used to classify a B cell FCXM are potential contributors to a T+B- FCXM result.ConclusionsOur analysis of 3073 FCXM, in addition to demonstrating that HLA antibodies directed at HLA-A, B or Cw locus are associated with a T+B- result, identified mechanisms for the surprising T+B- FCXM result.

Identification of biomarkers that assess posttransplant risk is needed to improve long-term outcomes following heart transplantation. The Clinical Trials in Organ Transplantation (CTOT)-05 protocol was an observational, multicenter, cohort study of 200 heart transplant recipients followed for the first posttransplant year. The primary endpoint was a composite of death, graft loss/retransplantation, biopsy-proven acute rejection (BPAR), and cardiac allograft vasculopathy (CAV) as defined by intravascular ultrasound (IVUS). We serially measured anti-HLA- and auto-antibodies, angiogenic proteins, peripheral blood allo-reactivity, and peripheral blood gene expression patterns. We correlated assay results and clinical characteristics with the composite endpoint and its components. The composite endpoint was associated with older donor allografts (p

The relationship between circulating pre-transplant immunoglobulin G (IgG) antibodies to donor human leukocyte antigen (HLA) -C locus determined antigens alone and acute rejection, kidney allograft function, and graft survival is not fully defined. Also, the impact of circulating pre-transplant IgG antibodies to donor HLA-C locus antigens alone on these outcomes has not been compared with the impact of circulating pre-transplant IgG antibodies to donor HLA-A or -B locus antigens. We conducted a retrospective review of records of 1252 kidney allograft recipients transplanted at our center between January 2010 and January 2016 to identify patients with circulating pre-transplant IgG antibodies directed at kidney donor HLA-A, -B, or -C locus determined antigens. Antibodies were detected and reported using the LABScreen Single Antigen Bead assay with microbeads coated with single HLA class I antigens. Pre-transplant and post-transplant data were collected and the graft outcomes of 16 kidney graft recipients with antibodies to HLA-C locus antigens were compared to the outcomes in 56 recipients with antibodies to HLA-A or -B locus determined antigens. The one-year acute rejection rate was 6% in those with donor-specific antibodies (DSA) to HLA-C locus antigens and 20% in those with DSA to HLA-A or -B locus antigens. The graft survival rate was 100% in those with DSA to HLA-C locus antigens and 95% in those with DSA to HLA-A or -B locus antigens. None of the numerical differences were statistically significant (p0.05). The presence of circulating pre-transplant IgG antibodies directed at kidney donor HLA-C locus antigens alone may not be associated with an increased risk of acute rejection or a decreased graft survival rate. Our observations support the concept that circulating pre-transplant IgG antibodies directed at kidney donor HLA-C locus antigens alone do not negatively impact kidney allograft outcomes and that the mean fluorescence intensities of the antibodies directed at HLA-C locus alone should not be used to list unacceptable HLA-C locus antigens for kidney allocation. A study with a larger cohort is needed to investigate our hypothesis.

CD4+ T helper (Th) cell subset generation in vivo requires T cell receptor activation and surface CD28 co-stimulation in the presence of one or more cytokines. Similarly, Th cells can be generated in vitro by activating naïve CD4+CD25- T cells with plate bound-anti-CD3 monoclonal antibody (mAb) (pbCD3) and soluble-anti-CD28 mAb (sCD28) in the presence of polarizing recombinant (r) cytokines and anti-cytokine mAbs. In comparison to in vitro CD4+CD25- T cells, memory CD4+CD25-CD45RO+ T cells have been shown to convert to Th9 cells more efficiently. Here, protocol for in vitro generation of human Th9 cells by activating CD4+CD25-CD45RO+ memory T cells with pbCD3 and sCD28 in the presence of polarizing recombinant interleukin-4 (rIL-4) and transforming growth factor (rTGF-β) is described.

CD4+ T helper (Th) cell subset generation in vivo requires T cell receptor activation and surface CD28 co-stimulation in the presence of one or more cytokines. Similarly, Th cells can be generated in vitro by activating naive CD4+CD25- T cells with plate bound-anti-CD3 monoclonal antibody (mAb) (pbCD3) and soluble-anti-CD28 mAb (sCD28) in the presence of polarizing recombinant (r) cytokines and anti-cytokine mAbs. In comparison to in vitro CD4+CD25- T cells, memory CD4+CD25-CD45RO+ T cells have been shown to convert to Th9 cells more efficiently. Here, protocol for in vitro generation of human Th9 cells by activating CD4+CD25-CD45RO+ memory T cells with pbCD3 and sCD28 in the presence of polarizing recombinant interleukin-4 (rIL-4) and transforming growth factor (rTGF-β) is described.

Purinergic signaling and associated ectonucleotidases, such as CD39 and CD73, have been implicated in the pathogenesis of inflammatory bowel disease (IBD). CD39 is known to be a Treg memory cell marker, and here we determine the phenotype and function of CD73+CD4+ T lymphocytes in patients with IBD. We describe elevated levels of CD73+CD4+ T cells in the peripheral blood and intestinal lamina propria of patients with active IBD. The functional phenotype of these CD73+CD4+ T cells was further determined by gene expression, ecto-enzymatic activity, and suppressive assays. Increased numbers of CD73+CD4+ T cells in the periphery and lamina propria were noted during active inflammation, which returned to baseline levels following anti-TNF treatment. Peripheral CD73+CD4+ T cells predominantly expressed CD45RO, and were enriched with IL-17A+ cells. The CD73+CD4+ cell population expressed higher levels of RORC, IL-17A, and TNF, and lower levels of FOXP3 and/or CD25, than CD73−CD4+ T cells. Expression of CD73 by peripheral CD4+ T cells was increased by TNF, and decreased by an anti-TNF monoclonal antibody (infliximab). In vitro, these peripheral CD73+CD4+ T cells did not suppress proliferation of CD25− effector cells, and expressed higher levels of pro-inflammatory markers. We conclude that the CD73+CD4+ T-cell population in patients with active IBD are enriched with cells with a T-helper type 17 phenotype, and could be used to monitor disease activity during treatment.

We have previously reported that peri-transplant conditioning leads to successful induction of renal allograft tolerance via the mixed chimerism approach in nonhuman primates (NHP) and humans. However, this strategy requires treatments beginning six days prior to transplantation, which limits its relevance only to living donor transplant recipients. To extend the clinical applicability of this approach, we developed a novel regimen “delayed tolerance,” with which the recipient initially undergoes organ transplantation with conventional immunosuppression, followed by conditioning and donor bone marrow transplantation (DBMT) at a later date. This approach might be likened to “planting flowers in a battle field.” That is, the recipient’s immunologic environment after organ transplantation is like a battlefield filled with hostile innate and adaptive immune-responses directed against donor antigeneic specificities. Implanting fragile donor hematopoietic progenitors into this environment and encouraging them to bloom in this vicious field requires special treatments. In our NHP studies recently published in The American Journal of Transplantation, we showed that such “delayed tolerance,” in fact, can be induced in NHP through the mixed chimerism approach, if specific modifications to overcome/avoid donor-specific memory T cell responses are provided. These modifications include adequate depletion of CD8 memory T cells and timing of donor bone marrow administration to minimize levels of pro-inflammatory cytokines. This article addendum will provide a short summary of the original paper with our additional insights and interpretations.

Aim Emerging data suggest that DSA development is a frequent complication post BKV replication, and most DSA are directed against donor HLA-DQ. In the current investigation, we quantified DQ epitope mismatches between the recipient and donor in individuals with BKVN diagnosis and investigated the association between DQ epitope load and development of DSA. Methods In this pilot study, using the most probable 4-digit HLA typings, we investigated epitope mismatches in individuals who developed DSA post BKVN diagnosis compared to those who did not and correlated them to DSA development and graft dysfunction defined as an increase in serum creatinine by > 0.5 mg/dL at 12 months post-BKVN diagnosis. We had SSOP based intermediate HLA typing available for recipients and donors in 13 recipients who developed BKVN confirmed by renal allograft biopsy. Of the 13 recipients, 2 were excluded: 1 for early graft loss and 1 who had zero DQ mismatches. None of the 11 recipients with BKVN diagnosis had a positive XM or DSA at time of transplantation. Results Time from kidney transplantation to BKVN diagnosis was 8.5 ± 5.1 months. Serum creatinine at time of diagnosis was 1.78 ± 0.56. Of the 11 with BKVN, 36% developed de novo DSA. All DSAs were directed against DQ mismatches. Time from BKVN diagnosis to DSA development was 5.5 ± 2.3 months. Table below lists the epitope mismatch number at each locus. Of the 4 patients with de novo DSA, 75% experienced graft dysfunction during the 12 months post BKVN and of the 7 patients without de novo DSA, 29% experienced graft dysfunction by 12 months. Our data demonstrated that the most frequent DQ epitope mismatches in those with de novo DSA were 52PL3, 45EV, 52PQ2, and 52PR. Conclusions In our pilot study, the epitope load was not different between BKVN patients who developed or did not develop de novo DSA. The most frequent target of de novo antibodies were DQ locus mismatched epitopes. Knowledge of epitope targets of de novo DSA in patients with BKVN may facilitate targeted therapies and improve kidney allograft outcomes. Download : Download high-res image (148KB) Download : Download full-size image

Aim Luminex Single Antigen bead assay (LSAB) derived mean fluorescence intensity (MFI) values of anti-HLA antibodies directed at the potential donor’s HLA are the primary parameter used in the virtual crossmatch to predict physical crossmatch (XM) outcome. We aimed to develop statistically validated LSAB MFI cutpoints for predicting CDC XM and FCXM results. Methods We leveraged the ASHI proficiency testing (PT) 80% consensus results of 7156 T FCXM, 6758 B FCXM, 2917 T CDC XM and 2233 B CDC XM as the reference results to investigate whether LSAB MFI of IgG anti-HLA antibodies predict validated physical XM results. LSAB MFI was determined in our laboratory using One Lambda Single Antigen HLA Class I and Class II beads. The 80% consensus results are from 8 consecutive challenges during 2013 to 2016, and 107 laboratories across USA tested sera and HLA typed cells distributed by ASHI, and reported the results to ASHI for assessment of the laboratory’s proficiency. Data analysis included: summing of MFI of IgG antibodies directed at HLA-A, B and C for investigating their association with T FCXM and T CDC XM results; summing of MFI of antibodies directed at HLA-A, B, C, DR, DQ and DP for investigating their association with B FCXM and B CDC XM results; assigning alternate ASHI challenges to the Discovery set and the Validation set; investigating the association between LSAB MFI and physical XM results by logistic regression analysis corrected for overdispersion; and identification of LSAB MFI cutpoint for maximizing the sum of sensitivity and specificity. LSAB MFI cutpoints derived from the Discovery set to predict FCXM and CDC XM results were investigated in an independent validation set. Results Table 1 demonstrates that LSAB MFI cutpoints from the Discovery set predicts XM outcomes in the Validation set (ROC AUC range from 0.974 to 0.999). Conclusions We have developed and validated LSAB MFI cutpoints for use in the virtual crossmatch to accurately predict physical T FCXM, B FCXM, T CDC XM and B CDC XM results.

Aim Interfering factors in sera may confound crossmatch (XM) results. Heat inactivation (HI), DTT and dialysis treatment of sera have been utilized to minimize these confounders. We investigated the impact of HI on donor T cell complement dependent cytotoxicity (CDC) XM (T Cell AHG XM) and donor B cell CDC XM (B Cell NIH XM). Methods We queried our electronic database for all donor CDC T cell and B cell XM performed during 2011–2017 in which potential kidney transplant recipient’s serum was tested in parallel with or without HI and using potential donor’s T cells and B cells as target cells. Results A total of 6295 living donor XMs and 15880 deceased donor XMs performed in our laboratory were reviewed. Table 1 shows the impact of HI on the 22175 consecutive XM results. 394 of the 6295 living donor T Cell AHG XMs were positive using neat sera and 303 were positive following HI at 63C for 6 min, a 23% conversion from positive to negative results. 698 of the 15880 deceased donor T Cell AHG XMs were positive using neat sera and 540 XMs were positive following HI, a 23% reduction. 1372 of the 6295 living donor B Cell NIH XMs were positive using neat sera and 539 were positive following HI, a 61% reduction. 3015 of the 15880 deceased donor B Cell NIH XMs were positive using neat sera and 842 XMs were positive following HI, a 72% reduction from a positive result to negative result. Conclusions A substantial percentage of XMs are converted from a positive CDC result to negative result and such conversion is more frequent with B cell XM compared to T cell XM. Transplant centers seldom transplant across a positive T cell CDC XM even when HI converts a positive result to a negative result. On the other hand, it is not uncommon to proceed with a kidney transplant when HI converts a positive B cell CDC to a negative CDC provided there are no other immune contraindications. There is an urgent need to establish the clinical significance of XMs converted from a positive to a negative result by heat inactivation.

We have previously demonstrated that biomarkers of inflammation and immune activity detected within intraoperative renal transplant allograft biopsies are linked to adverse short-term post-transplantation clinical outcomes. Now we provide a post hoc analysis of our earlier data in the light of longer clinical follow-up. A total of 75 consecutively performed renal allografts were analyzed for gene expression of proinflammatory molecules, inflammation-induced adhesion molecules, and antiapoptotic genes expressed 15 minutes after vascular reperfusion to determine whether this analysis can aid in predicting long-term quality of renal function, proteinuria, graft loss, and death-censored graft. We have built predictive models for proteinuria (area under the curve = 0.859, p = 0.0001) and graft loss (area under the curve = 0.724, p = 0.027) 2 years post-transplantation using clinical variables in combination with intragraft gene expression data of tumor necrosis factor-alpha, interleukin-6, CD40, CD3, and tumor necrosis factor-alpha, Bcl-2, and interferon-gamma, respectively. This post hoc analysis demonstrates that hypothesis-driven, targeted polymerase chain reaction profiling of gene expression in the donor kidney at the time of engraftment can predict 2-year post-transplantation clinical outcomes.

Within the immune system there is an exquisite ability to discriminate between "self" and "non-self" that is orchestrated by T lymphocytes. Discriminatory pathways guide differentiation of these lymphocytes into either regulatory (Treg) or effector (Teff) T cells, influenced by cues from the naïve T cell's immediate micro-environment as it responds to cognate antigen. Reciprocal pathways may lead to commitment of naïve T cells into either the protective tolerance-promoting Treg, or to the pro-inflammatory Th17 effector phenotype. Primary activation of CD4(+) lymphocytes stimulates their release of leukemia inhibitory factor (LIF), and Treg continue to release LIF in response to antigen, implying a role for LIF in tolerance. In contrast, interleukin- 6 (IL-6), although very closely related to LIF, promotes maturation of Th17 cells. Here we show that LIF and IL-6 behave as polar opposites in promoting commitment to the Treg and Th17 lineages. Unlike IL6, LIF supported expression of Foxp3, the Treg lineage transcription factor, and LIF opposed IL6 by suppressing IL-6-induced IL-17A protein release. In striking contrast, we found that IL6 effectively inhibited LIF signalling, repressing transcription of the LIF receptor gp190, and strongly inducing axotrophin/MARCH-7, a novel E3 ubitquitin ligase that we discovered to be active in degradation of gp190 protein. In vivo, anti-LIF treatment reduced donor-specific Treg in recipients of foreign spleen cells. Conversely, a single dose of biodegradable LIF nanoparticles, targeted to CD4, successfully manipulated the LIF/IL6 axis towards development of donor-specific Foxp3(+) Treg. The implications for therapy are profound, harnessing endogenous immune regulation by paracrine delivery of LIF to CD4(+) cells in vivo.

In nonobese diabetic (NOD) mice with overt new-onset type 1 diabetes mellitus (T1DM), short-term treatment with a “triple-therapy” regimen [rapamycin plus agonist IL-2-related and antagonist-type, mutant IL-15-related Ig fusion proteins (IL-2.Ig and mutIL-15.Ig)] halts autoimmune destruction of insulin-producing beta cells and restores both euglycemia and immune tolerance to beta cells. Increases in the mass of insulin-producing beta cells or circulating insulin levels were not linked to the restoration of euglycemia. Instead, the restoration of euglycemia was linked to relief from an inflammatory state that impaired the host's response to insulin. Both restoration of immune tolerance to beta cells and relief from the adverse metabolic effects of an inflammatory state in insulin-sensitive tissues appear essential for permanent restoration of normoglycemia in this T1DM model. Thus, this triple-therapy regimen, possessing both tolerance-inducing and select antiinflammatory properties, may represent a prototype for therapies able to restore euglycemia and self-tolerance in T1DM.

Experimental animal models for autoimmunity have demonstrated the existence and crucial role of CD4(+)CD25(+) T regulatory (Tr) cells in suppressing autoreactive T cells and promoting peripheral tolerance. Recent in vitro functional studies showed that Tr cells are enriched in the CD25(high) cell population among CD4(+) T cells, and that they totally inhibit proliferation and cytokine secretion by CD4(+) T cells. It is not yet known if circulating Tr cells are involved in multiple sclerosis (MS). This study was done firstly to determine whether alterations of the CD4 (+) CD25(high) T cells occur in MS, examining their frequencies. As it was reported that the suppressive activity of CD4(+)CD25(+) Tr cells is mainly through cell surface contact pathway, we secondly analyzed the expression of the functionally important cell surface molecules of CD4(+)CD25(high) Tr cells. Two- or three-colour flow cytometry was used to identify and quantify CD4(+)CD25(+) Tr cells and CD4(+)CD25(high) Tr cells among blood CD4(+) T cells in MS patients without treatment vs. patients treated with either interferon-beta (IFN-beta) or glatiramer acetate (GA) or IFN-beta + GA in combination vs. healthy controls (HC). Expression of functionally important surface molecules CD45RO, CD69, CD95, HLA-DR, and intracellular CTLA-4 and IL-10 production by CD4(+)CD25(high) Tr cells were investigated. CD4(+)CD25(+) T cells constituted around 6% of CD4(+)T cells in all MS patient groups, and 7% in HC. There were also no changes in the proportions of CD4(+)CD25(+) Tr cells and CD4(+)CD25(high) Tr cells in a longitudinal follow-up of MS patients before and during IFN-beta treatment. Frequencies of circulating CD4(+)CD25(high)Tr cells among CD4(+) T cells were also similar and their surface or intracellular molecular expression did not vary in MS patients, irrespective of treatment, compared to HC. This study suggests that levels of circulating CD4(+)CD25(+) Tr cells and CD4(+)CD25(high) Tr cells are not altered in MS, and are unaffected by substances currently used to modulate the disease.

Magnetic resonance imaging (MRI) remains the most valuable tool for monitoring disease activity and progression in patients with multiple sclerosis (MS), a chronic demyelinating disease of the central nervous system (CNS) with presumably autoimmune etiology. Chemokine receptors have been implicated in MS as key molecules directing inflammatory cells into the CNS. Regulatory (CD4+CD25+) T cells (Tr cells) are important in suppressing autoimmunity, and their absolute or functional deficit could be expected in MS. In the present study, venous blood was obtained from MS patients concurrent with MRI examination of the brain, and expression of chemokine receptors CCR1, CCR2, CCR5, CXCR3 and CXCR4 by CD4 T cells and monocytes, proportions of Tr cells, as well as expression of CD45RO, CD95, CTLA-4, HLA-DR and interleukin (IL)-10 by Tr cells and non-Tr (CD25-) CD4 T cells was analyzed by flow cytometry. Surface expression of CXCR3 by CD4 T cells was downregulated in the group of patients with high lesion load (LL) on T2-weighted images and gadolinium (Gd)-enhancing lesions on T1-weighted images, compared to the group with high LL and no Gd-enhancing lesions, and to the group with low LL, suggesting internalization of CXCR3 due to the release of its chemokine ligand (IP-10/CXCL10) from active MS lesions. Proportions of Tr cells amongst all CD4 T cells, and expression of IL-10 by Tr cells were increased in the patients with high LL and Gd-enhancing lesions. These results suggest that there is correlation between MRI parameters, chemokine receptor expression and the status of circulating Tr cells in MS, but further studies need to discriminate between pathogenetically relevant and bystander phenomena.

Aim Luminex Single Antigen bead assays (LSAB) are robust for detecting and identifying IgG anti-HLA antibodies, and the LSAB mean fluorescence intensity (MFI) is currently used to list unacceptable antigens and to predict physical crossmatch (XM) results. Concerns exist regarding the use of MFI as the criterion because MFI thresholds for accurately predicting physical XM results are far from established. We aimed to address this unmet need. Methods We leveraged the XM results from ASHI Proficiency Testing (PTs) to investigate whether LSAB MFI cutpoints are predictive of physical XM results. ASHI graded (⩾80% consensus) XM results from 8 consecutive ASHI PTs in which up to 30 labs tested 40 sera and 16 cells distributed by ASHI were used to examine whether LSAB MFI data we generated in the same PTs predict physical XM results. Cumulative MFIs of DSA directed at HLA-A, B, and C were tested for their ability to predict T cell Flow XM and T cell AHG XM results; cumulative MFIs of DSA directed at HLA-A, B, C, DRB1, DRB3/4/5, DQB and DP were tested for their ability to predict B cell Flow XM and B cell CDC XM results. Results Our data analysis identified that cumulative MFI of DSA less than 6000 directed at HLA-class I antigens predicted T cell Flow XM results with a 100% negative predictive value (NPV) and MFI > 7000 predicted T cell Flow XM results with a 100% positive predictive value (PPV) (Chi-Square for trend, X 2 = 70, df = 1, P 8000 predicted B Flow XM result with a 100% PPV (X 2 ; =; 67, df = 1, P Download high-res image (343KB) Download full-size image Conclusions Our data demonstrating the feasibility of developing LSAB MFI cutpoints for the accurate prediction of T cell Flow XM, B cell Flow XM, T cell AHG XM, and B cell CDC XM results advance a strategy for the interpretation of virtual crossmatches and the prediction of physical XM results.

Aim LSAB mean fluorescence intensity (MFI) is often used to predict physical FCXM results, and LSAB MFI cutpoints are used to list unacceptable antigens. We therefore determined the accuracy of LSAB assay MFI cutpoint used to score the LSAB assay as positive or negative in foretelling FCXM results. Methods One hundred and forty-six pre-transplant sera from 146 patients with end stage renal disease were tested against potential kidney donor’s T cells and B cells using a 3-color flow cytometry clinical protocol; the same 146 sera were tested using LSAB assay and the cumulative MFI directed at donor HLA class I and/or II antigens were determined. A T cell FCXM with a median channel shift (MCS) ≥ 40 with the patients serum vs. negative control serum was classified as a positive test; a B cell FCXM with MCS ≥ 50 with the patients serum vs. negative control serum was classified as a positive test. In the LSAB assay, MFI > 2000 directed at donor HLA antigens was scored as positive DSA. These cutpoints are our laboratory’s cutpoints for both ASHI proficiency testing and for clinical use. Results Statistical analysis demonstrated a significant association between LSAB results and FXCM results (Table 1). LSAB assay results predicted T Cell FCXM results with a sensitivity of 70% and a specificity of 100% (Fischers Exact Test, P 0.0001, Positive Predictive Value (PPV = 100%, Negative Predictive Value (NPV) = 91%). LSAB assay results also predicted B Cell FCXM results with a sensitivity of 86% and a specificity of 72% (Fischers Exact Test, P 0.0001, PPV = 62%, NPV = 91%) (Table 1). In a subset of 31 patients, both donor FCXM and auto FCXM results were available and 17 of 31 donor positive FCXM were also auto B cell FCXM positive (17/17) and auto T cell FCXM positive (1/17). Conclusions LSAB assay results are significantly associated with FCXM results. Auto B cell FCXM and high resolution HLA typing should further refine the relationship between these two high sensitive assays. Download high-res image (138KB) Download full-size image

Effective means to identify anti-donor immune activity before the transplant organ is damaged and rejected has been an important goal in transplantation research. Development of sensitive and non-invasive diagnostic methods that probe the immune status of the recipient as well as the resilience of the donor organ should enable personalized application of immunosuppressive drugs. With a non-invasive biomarker for rejection, it should be possible to selectively treat the patients that are rejecting the graft and wean the tolerant patients from immunosuppression. Although A20 is also expressed by activated CD4+ T cells and CD8+ T cells, its expression by mouse tubular cells has been shown to play an important role in protecting allografts from ischemia/reperfusion (I/R) injury and rejection. Using quantitative (real-time) reverse transcriptase polymerase chain reaction (qt-RT-PCR), we showed that expression levels of A20, heme oxygenase (HO)-1, other anti-apoptotic molecules, granzyme-B (GZMB), perforin (PRF1), CD3 and other immune molecules in renal transplant biopsies, urinary cells and peripheral blood cells are predictive of transplantation outcomes. Measuring A20 at mRNA and protein levels has the potentiality to be diagnostic and prognostic of transplantation outcomes and thereby help in timely therapeutic interventions to prolong graft life.

Effective means to identify anti-donor immune activity before the transplant organ is damaged and rejected has been an important goal in transplantation research. Development of sensitive and non-invasive diagnostic methods that probe the immune status of the recipient as well as the resilience of the donor organ should enable personalized application of immunosuppressive drugs. With a non-invasive biomarker for rejection, it should be possible to selectively treat the patients that are rejecting the graft and wean the tolerant patients from immunosuppression. Although A20 is also expressed by activated CD4+ T cells and CD8+ T cells, its expression by mouse tubular cells has been shown to play an important role in protecting allografts from ischemia/reperfusion (I/R) injury and rejection. Using quantitative (real-time) reverse transcriptase polymerase chain reaction (qt-RT-PCR), we showed that expression levels of A20, hemeoxygenase (HO)-1, other anti-apoptotic molecules, granzyme-B (GZMB), perforin (PRF1), CD3 and other immune molecules in renal transplant biopsies, urinary cells and peripheral blood cells are predictive of transplantation outcomes. Measuring A20 at mRNA and protein levels has the potentiality to be diagnostic and prognostic of transplantation outcomes and thereby help in timely therapeutic interventions to prolong graft life.

An experimental and theoretical study of T cell differentiation in response to mixed-input conditions reveals that cells can tune between Th1 and Th2 states through a continuum of mixed phenotypes., Cell differentiation is typically directed by external signals that drive opposing regulatory pathways. Studying differentiation under polarizing conditions, with only one input signal provided, is limited in its ability to resolve the logic of interactions between opposing pathways. Dissection of this logic can be facilitated by mapping the system's response to mixtures of input signals, which are expected to occur in vivo, where cells are simultaneously exposed to various signals with potentially opposing effects. Here, we systematically map the response of naïve T cells to mixtures of signals driving differentiation into the Th1 and Th2 lineages. We characterize cell state at the single cell level by measuring levels of the two lineage-specific transcription factors (T-bet and GATA3) and two lineage characteristic cytokines (IFN-γ and IL-4) that are driven by these transcription regulators. We find a continuum of mixed phenotypes in which individual cells co-express the two lineage-specific master regulators at levels that gradually depend on levels of the two input signals. Using mathematical modeling we show that such tunable mixed phenotype arises if autoregulatory positive feedback loops in the gene network regulating this process are gradual and dominant over cross-pathway inhibition. We also find that expression of the lineage-specific cytokines follows two independent stochastic processes that are biased by expression levels of the master regulators. Thus, cytokine expression is highly heterogeneous under mixed conditions, with subpopulations of cells expressing only IFN-γ, only IL-4, both cytokines, or neither. The fraction of cells in each of these subpopulations changes gradually with input conditions, reproducing the continuous internal state at the cell population level. These results suggest a differentiation scheme in which cells reflect uncertainty through a continuously tuneable mixed phenotype combined with a biased stochastic decision rather than a binary phenotype with a deterministic decision., Author Summary During cell differentiation, progenitor cells respond to external signals that drive the expression of genes that are characteristic of the differentiated cell states. This process is controlled by gene regulatory networks that typically involve positive autoregulation and cross-inhibition between master regulators of the two differentiated states. Mapping the system's response to mixtures of external signals can help us to understand the operational logic of these binary cell fate decisions. Here, we study differentiation of CD4+ T cells into Th1 and Th2 lineages under mixed-input conditions, at the single cell level. We reveal that cell state is not restricted to a small number of well-defined phenotypes, but rather tunes through a continuum of mixed-phenotype states in which levels of lineage-specifying transcription factors gradually change with the levels of the two inputs. Using mathematical modeling we establish the conditions under which the system has one stable steady state that continuously tunes in response to changes in levels of the inputs. Results of this model qualitatively explain our experimental observations. We further characterize expression patterns of downstream lineage-specific genes—cytokines that are driven by the two master regulators upon cell re-stimulation. We find a highly heterogeneous population with cells expressing either one of the cytokines, both cytokines, or neither. Of note, the fraction of cells in these subpopulations continuously tunes with input levels, thus reproducing a tunable state at the cell population level. Our results can be explained by a two-stage scheme in which the gene regulatory network is responsible for a continuously tunable cell state, which is translated into a heterogeneous cytokine expression pattern through uncorrelated and biased stochastic processes.

The predominant anti-HLA IgG subclass during antibody mediated rejection episodes is IgG1. Class I DSA IgG subclasses respond to single cycle of bortezomib-based therapy more frequently as compared to Class II DSA IgG subclasses. High titer Class II DSA IgG subclasses are less likely to respond to single cycle bortezomib-based therapy. Future studies are needed to determine if the response to bortezomib-based therapy is dependent on the type of DSA and/or the strength of the DSA at the time of antibody mediated rejection episode.

Purinergic signaling and associated ectonucleotidases, such as CD39 and CD73, have been implicated in the pathogenesis of inflammatory bowel disease (IBD). CD39 is known to be a Treg memory cell marker, and here we determine the phenotype and function of CD73(+) CD4(+) T lymphocytes in patients with IBD. We describe elevated levels of CD73(+) CD4(+) T cells in the peripheral blood and intestinal lamina propria of patients with active IBD. The functional phenotype of these CD73(+) CD4(+) T cells was further determined by gene expression, ecto-enzymatic activity, and suppressive assays. Increased numbers of CD73(+) CD4(+) T cells in the periphery and lamina propria were noted during active inflammation, which returned to baseline levels following anti-TNF treatment. Peripheral CD73(+) CD4(+) T cells predominantly expressed CD45RO, and were enriched with IL-17A(+) cells. The CD73(+) CD4(+) cell population expressed higher levels of RORC, IL-17A, and TNF, and lower levels of FOXP3 and/or CD25, than CD73(-) CD4(+) T cells. Expression of CD73 by peripheral CD4(+) T cells was increased by TNF, and decreased by an anti-TNF monoclonal antibody (infliximab). In vitro, these peripheral CD73(+) CD4(+) T cells did not suppress proliferation of CD25(-) effector cells, and expressed higher levels of pro-inflammatory markers. We conclude that the CD73(+) CD4(+) T-cell population in patients with active IBD are enriched with cells with a T-helper type 17 phenotype, and could be used to monitor disease activity during treatment.

The promise of islet cell transplantation cannot be fully realized in the absence of improvements in engraftment of resilient islets. The marginal mass of islets surviving the serial peritransplant insults may lead to exhaustion and thereby contribute to an unacceptably high rate of intermediate and long-term graft loss. Hence, we have studied the effects of treatment with alpha 1-antitrypsin (AAT) in a syngeneic nonautoimmune islet graft model. A marginal number of syngeneic mouse islets were transplanted into nonautoimmune diabetic hosts and islet function was analyzed in control and AAT treated hosts. In untreated controls, marginal mass islet transplants did not restore euglycemia. Outcomes were dramatically improved by short-term AAT treatment. Transcriptional profiling identified 1,184 differentially expressed transcripts in AAT-treated hosts at 3 d posttransplantation. Systems-biology–based analysis revealed AAT down-regulated regulatory hubs formed by inflammation-related molecules (e.g., TNF-α, NF-κB). The conclusions yielded by the systems-biology analysis were rigorously confirmed by QRT-PCR and immunohistology. These data suggest that short-term AAT treatment of human islet transplant recipients may be worthy of a clinical trial.

Inflammation induces the NF-κB dependent protein A20 in human renal proximal tubular epithelial cells (RPTEC), which secondarily contains inflammation by shutting down NF-κB activation. We surmised that inducing A20 without engaging the pro-inflammatory arm of NF-κB could improve outcomes in kidney disease. We showed that hepatocyte growth factor (HGF) increases A20 mRNA and protein levels in RPTEC without causing inflammation. Upregulation of A20 by HGF was NF-κB/RelA dependent as it was abolished by overexpressing IκBα or silencing p65/RelA. Unlike TNFα, HGF caused minimal IκBα and p65/RelA phosphorylation, with moderate IκBα degradation. Upstream, HGF led to robust and sustained AKT activation, which was required for p65 phosphorylation and A20 upregulation. While HGF treatment of RPTEC significantly increased A20 mRNA, it failed to induce NF-κB dependent, pro-inflammatory MCP-1, VCAM-1, and ICAM-1 mRNA. This indicates that HGF preferentially upregulates protective (A20) over pro-inflammatory NF-κB dependent genes. Upregulation of A20 supported the anti-inflammatory effects of HGF in RPTEC. HGF pretreatment significantly attenuated TNFα-mediated increase of ICAM-1, a finding partially reversed by silencing A20. In conclusion, this is the first demonstration that HGF activates an AKT-p65/RelA pathway to preferentially induce A20 but not inflammatory molecules. This could be highly desirable in acute and chronic renal injury where A20-based anti-inflammatory therapies are beneficial.

Background Previously, we have demonstrated that short-term treatment of new onset diabetic Non-obese diabetic (NOD) mice, mice that are afflicted with both type 1 (T1D) and type 2 (T2D) diabetes with either Power Mix (PM) regimen or alpha1 antitrypsin (AAT) permanently restores euglycemia, immune tolerance to self-islets and normal insulin signaling. Methodology and principal findings To search for relevant therapeutic targets, we have applied genome wide transcriptional profiling and systems biology oriented bioinformatics analysis to examine the impact of the PM and AAT regimens upon pancreatic lymph node (PLN) and fat, a crucial tissue for insulin dependent glucose disposal, in new onset diabetic non-obese diabetic (NOD) mice. Systems biology analysis identified tumor necrosis factor alpha (TNF-α) as the top focus gene hub, as determined by the highest degree of connectivity, in both tissues. In PLNs and fat, TNF-α interacted with 53% and 32% of genes, respectively, associated with reversal of diabetes by previous treatments and was thereby selected as a therapeutic target. Short-term anti-TNF-α treatment ablated a T cell-rich islet-invasive and beta cell-destructive process, thereby enhancing beta cell viability. Indeed anti-TNF-α treatment induces immune tolerance selective to syngeneic beta cells. In addition to these curative effects on T1D anti-TNF-α treatment restored in vivo insulin signaling resulting in restoration of insulin sensitivity. Conclusions In short, our molecular analysis suggested that PM and AAT both may act in part by quenching a detrimental TNF-α dependent effect in both fat and PLNs. Indeed, short-term anti-TNF-α mAb treatment restored enduring euglycemia, self-tolerance, and normal insulin signaling.

It has been reported that rapamycin (RPM) can induce de novo conversion of the conventional CD4+Foxp3-T cells into CD4+Foxp3+ regulatory T cells (iTregs) in transplantation setting. It is not clear whether RPM can similarly generate suppressor CD8+ T cells to facilitate graft acceptance. In this study, we investigated the ability of short-term RPM treatment in promoting long-term acceptance (LTA) of MHC-mismatched skin allografts by generating a CD8+ suppressor T-cell population. We found that CD4 knockout (KO) mice (in C57BL/6 background, H-2b) can promptly reject DBA/2 (H-2d) skin allografts with mean survival time (MST) being 13 days ( p < 0.01). However, a short course RPM treatment in these animals induced LTA with graft MST longer than 100 days. Adoptive transfer of CD8+ T cells from LTA group into recombination-activating gene 1 (Rag-1)-deficient mice provided donor-specific protection of DBA/2 skin grafts against cotransferred conventional CD8+ T cells. Functionally active immunoregulatory CD8+ T cells also resided in donor skin allografts. Eighteen percent of CD8+ suppressor T cells expressed CD28 as measured by flow cytometry, and produced reduced levels of IFN-γ, IL-2, and IL-10 in comparison to CD8+ effector T cells as measured by ELISA. It is unlikely that CD8+ suppressor T cells mediated graft protection via IL-10, as IL-10/Fc fusion protein impaired RPM-induced LTA in CD4 KO mice. Our data supported the notion that RPM-induced suppressor CD8+ T cells home to the allograft and exert donor-specific graft protection.

Purpose Resistance to antiangiogenic therapy is an important clinical problem. We examined whether resistance occurs at least in part via reversible, physiologic changes in the tumor, or results solely from stable genetic changes in resistant tumor cells. Experimental Design Mice bearing two human RCC xenografts were treated with sorafenib until they acquired resistance. Resistant 786-O cells were harvested and reimplanted into naive mice. Mice bearing resistant A498 cells were subjected to a 1 week treatment break. Sorafenib was then again administered to both sets of mice. Tumor growth patterns, gene expression, viability, blood vessel density, and perfusion were serially assessed in treated vs control mice. Results Despite prior resistance, reimplanted 786-O tumors maintained their ability to stabilize on sorafenib in sequential reimplantation steps. A transcriptome profile of the tumors revealed that the gene expression profile of tumors upon reimplantation reapproximated that of the untreated tumors and was distinct from tumors exhibiting resistance to sorafenib. In A498 tumors, revascularization was noted with resistance and cessation of sorafenib therapy and tumor perfusion was reduced and tumor cell necrosis enhanced with re-exposure to sorafenib. Conclusions In two RCC cell lines, resistance to sorafenib appears to be reversible. These results support the hypothesis that resistance to VEGF pathway therapy is not solely the result of a permanent genetic change in the tumor or selection of resistant clones, but rather is due to a great extent to reversible changes that likely occur in the tumor and/or its microenvironment.

We have shown that CD39 and CD73 are co-expressed on the surface of murine CD4+Foxp3+ regulatory T cells (Treg) and generate extracellular adenosine, contributing to Treg immunosuppressive activity. We now describe that CD39, independently of CD73, is expressed by a subset of blood derived human CD4+CD25+CD127lo T regulatory cells (Treg), defined by robust expression of Foxp3. A further distinct population of CD4+CD39+ T lymphocytes can be identified, which do not express CD25 and FoxP3 and exhibit the memory effector cellular phenotype. Differential expression of CD25 and CD39 on circulating CD4+ T cells distinguishes between Treg and pathogenic cellular populations that secrete pro-inflammatory cytokines such as IFNγ and IL-17. These latter cell populations are increased, with a concomitant decrease in the CD4+CD25+CD39+ Tregs, in the peripheral blood of patients with renal allograft rejection. We conclude that the ectonucleotidase CD39 is a useful and dynamic lymphocytes surface marker that can be used to identify different peripheral blood T cell populations to allow tracking of these in health and disease, as in renal allograft rejection.

A new study identifies the bone marrow haematopoietic stem cell (HSC) niche — a specialized microenvironment where stem cells reside — as an immune privileged site. This property is known to exist in the testis, ovary and hair follicle but has not been universally demonstrated in all stem cell niches. High-resolution in vivo imaging shows the accumulation of regulatory T cells in the HSC niche, enabling transplanted allo-HSCs to escape from allogeneic rejection. As well as supporting stem-cell function, the niche may provide a relative sanctuary from immune attack that could extend to malignant cells in some instances. Stem cells reside in a specialized regulatory microenvironment or niche1,2, where they receive appropriate support for maintaining self-renewal and multi-lineage differentiation capacity1,2,3. The niche may also protect stem cells from environmental insults3 including cytotoxic chemotherapy and perhaps pathogenic immunity4. The testis, hair follicle and placenta are all sites of residence for stem cells and are immune-suppressive environments, called immune-privileged sites, where multiple mechanisms cooperate to prevent immune attack, even enabling prolonged survival of foreign allografts without immunosuppression4. We sought to determine if somatic stem-cell niches more broadly are immune-privileged sites by examining the haematopoietic stem/progenitor cell (HSPC) niche1,2,5,6,7 in the bone marrow, a site where immune reactivity exists8,9. We observed persistence of HSPCs from allogeneic donor mice (allo-HSPCs) in non-irradiated recipient mice for 30 days without immunosuppression with the same survival frequency compared to syngeneic HSPCs. These HSPCs were lost after the depletion of FoxP3 regulatory T (Treg) cells. High-resolution in vivo imaging over time demonstrated marked co-localization of HSPCs with Treg cells that accumulated on the endosteal surface in the calvarial and trabecular bone marrow. Treg cells seem to participate in creating a localized zone where HSPCs reside and where Treg cells are necessary for allo-HSPC persistence. In addition to processes supporting stem-cell function, the niche will provide a relative sanctuary from immune attack.

Foxp3(+) regulatory T cells (Tregs) express both ectoenzymes CD39 and CD73, which in tandem hydrolyze pericellular ATP into adenosine, an immunoinhibitory molecule that contributes to Treg suppressive function. Using Foxp3GFP knockin mice, we noted that the mouse CD4(+)CD39(+) T-cell pool contains two roughly equal size Foxp3(+) and Foxp3(-) populations. While Foxp3(+)CD39(+) cells are CD73(bright) and are the bone fide Tregs, Foxp3(-)CD39(+) cells do not have suppressive activity and are CD44(+)CD62L(-)CD25(-)CD73(dim/-), exhibiting memory cell phenotype. Functionally, CD39 expression on memory and Treg cells confers protection against ATP-induced apoptosis. Compared with Foxp3(-)CD39(-) naïve T cells, Foxp3(-)CD39(+) cells freshly isolated from non-immunized mice express at rest significantly higher levels of mRNA for T-helper lineage-specific cytokines IFN-gamma (Th1), IL-4/IL-10 (Th2), IL-17A/F (Th17), as well as pro-inflammatory cytokines, and rapidly secrete these cytokines upon stimulation. Moreover, the presence of Foxp3(-)CD39(+) cells inhibits TGF-beta induction of Foxp3 in Foxp3(-)CD39(-) cells. Furthermore, when transferred in vivo, Foxp3(-)CD39(+) cells rejected MHC-mismatched skin allografts in a much faster tempo than Foxp3(-)CD39(-) cells. Thus, besides Tregs, CD39 is also expressed on pre-existing memory T cells of Th1-, Th2- and Th17-types with heightened alloreactivity.

Interleukin-31, produced mainly by activated CD4(+) T cells, is a newly discovered member of the gp130/IL-6 cytokine family. Unlike all the other family members, IL-31 does not engage gp130. Its receptor heterodimer consists of a unique gp130-like receptor chain IL-31RA, and the receptor subunit OSMRbeta that is shared with another family member oncostatin M (OSM). Binding of IL-31 to its receptor activates Jak/STAT, PI3K/AKT and MAPK pathways. IL-31 acts on a broad range of immune- and non-immune cells and therefore possesses potential pleiotropic physiological functions, including regulating hematopoiesis and immune response, causing inflammatory bowel disease, airway hypersensitivity and dermatitis. This review summarizes the recent findings on the biological characterization and physiological roles of IL-31 and its receptors.

Invasive insulitis is a destructive T cell-dependent autoimmune process directed against insulin-producing beta cells that is central to the pathogenesis of type 1 diabetes mellitus (T1DM) in humans and the clinically relevant nonobese diabetic (NOD) mouse model. Few therapies have succeeded in restoring long-term, drug-free euglycemia and immune tolerance to beta cells in overtly diabetic NOD mice, and none have demonstrably enabled enlargement of the functional beta cell mass. Recent studies have emphasized the impact of inflammatory cytokines on the commitment of antigen-activated T cells to various effector or regulatory T cell phenotypes and insulin resistance and defective insulin signaling. Hence, we tested the hypothesis that inflammatory mechanisms trigger insulitis, insulin resistance, faulty insulin signaling, and the loss of immune tolerance to islets. We demonstrate that treatment with alpha1-antitrypsin (AAT), an agent that dampens inflammation, does not directly inhibit T cell activation, ablates invasive insulitis, and restores euglycemia, immune tolerance to beta cells, normal insulin signaling, and insulin responsiveness in NOD mice with recent-onset T1DM through favorable changes in the inflammation milieu. Indeed, the functional mass of beta cells expands in AAT-treated diabetic NOD mice.

4-1BB ligand (4-1BBL; CD137L) is a member of the tumour necrosis factor superfamily expressed primarily on antigen presenting cells such as B cells, macrophages and dendritic cells. Its engagement with the receptor 4-1BB (CD137) has been shown to promote T-cell activation and regulate proliferation and survival of T cells. The role of the costimulatory molecule in multiple sclerosis (MS) remains unclear. In this study, the expression of 4-1BBL and soluble 4-1BBL (s4-1BBL) protein levels were analysed in peripheral blood of MS patients. Compared with healthy controls, MS patients had an increase in both plasma s4-1BBL protein levels and expression of 4-1BBL in CD14(+) monocytes. In contrast, myelin basic protein-reactive T-cell proliferation was not found to be inhibited by the use of an anti-4-1BBL antibody. The elevated s4-1BBL protein levels in the MS patients may function as a self-regulatory mechanism of 4-1BB/4-1BBL interaction and costimulation.

Magnetic resonance imaging (MRI) allows examining inflammation and central nervous system (CNS) tissue damage in patients suffering from multiple sclerosis (MS), a demyelinating disease of the CNS. Using real-time PCR, we quantified mRNA levels of apoptosis regulators CD95, CD95 ligand, caspase-8, -10 and cellular FLICE-inhibitory protein (cFLIP), and cytokines IL-10 and TNF-alpha in blood mononuclear cells of MS patients at the time of MRI examination. Patients with detectable gadolinium-enhancing lesions had lower expression of CD95 and caspase-8 (P0.05). Lesion load and brain atrophy did not correlate with expression levels of any of the target molecules studied. Disease duration correlated positively with both FLIP/caspase-8 and CD95/CD3 ratios (P0.05). These results support the notion that the CD95-dependent pathway plays a complex role in the regulation of survival of activated immune cells in MS.

CD4(+)CD25(high) T regulatory (Tr) cells, representing high IL-2 receptor alpha chain expressing cells, have been shown to inhibit proliferation and cytokine secretion by CD4(+) T cells that are assumed to represent important effector cells in auto-aggressive immunity. Tr cells may therefore be considered of importance in the pathogenesis of multiple sclerosis (MS). Glatiramer acetate (GA; Copaxone) is approved as a disease-modulating agent that ameliorates the course of MS. The goal of this study was to examine in vitro effects of GA on Tr cells from MS patients subgrouped according to treatment without or with disease-modulating drugs, and healthy controls (HC). Three-colour flow cytometry was used to investigate in vitro influence of GA, and of the encephalitogenic myelin basic protein (MBP) peptide 83-89 as control, on the blood Tr cell proportion and on their functionally important cell surface molecules CD45RO, CD69, CD95 and HLA-DR, and on intracellular CTLA-4 and IL-10. Irrespective of exposure to GA or MBP((83-99)), levels of blood Tr cells expressing HLA-DR remained low in untreated MS patients and HC compared to the three treated MS patient groups. In vitro exposure to GA resulted in elevated levels of IL-10 producing Tr cells in all MS patient groups irrespective of receiving treatment as well as in HC. Exposure to GA or MBP((83-99)) had no effects on levels of Tr cells expressing other above-mentioned molecules. We conclude that GA induces elevated IL-10 production by Tr cells that is uniform and independent of ongoing MS treatment with IFN-beta or GA or IFN-beta+GA.

Background In flow cytometry crossmatches (FCXM), sera from AB blood group male donors with none or minimal anti-HLA IgG antibodies are used as the negative control serum (NCS). As NCS can give higher median channel fluorescence (MCF) on certain donor cells, it is advised to use cells treated with phosphate buffer saline (PBS) as an additional negative control for comparison. Aim We examined whether using PBS or NCS as the negative control and subsequent staining with FITC-goat-anti-human IgG polyclonal antibody (g α IgG pAb; Jackson Immunoresearch Laboratory; Cat# 109-096-098) or FITC-mouse-anti-human IgG monoclonal antibody (m α IgG mAb; BD biosciences; Cat# 555786; Clone: G18-145) impacts FCXM results. Methodology Peripheral blood ( N = 5) and serum ( N = 5) were collected from human subjects. Peripheral blood mononuclear cells (PBMC) were isolated by Ficoll density gradient separation. In 96 well V-bottom plate, 300,000 PBMC were incubated with 30 μl of serum for 20 min at 22 °C, washed and stained with 10 μl each of anti-CD3 PerCP and of anti-CD19 PE, and with either 10 μl (1:100 diluted) of FITC-gαIgG pAb or 60 μl of FITC-mαIgG mAb for 30 min at 4 °C. The cells were washed and flow cytometry was performed using BD FACS Canto II instrument and the data was analyzed using the DiVa software. A channel shift of >40 from the NCS for T cell and a shift of >50 for B cells is considered positive here. Results PBMCs treated with PBS gave positive B cell channel shift when stained with g α IgG pAb, but not when stained with m α IgG mAb ( Fig. 1 ). Unlike m α IgG mAb, the g α IgG pAb seems to bind non-specifically to B cells in the absence of human serum. Conclusion This observation cautions that in a clinical FCXM testing, the non use of NCS can lead to a false positive FCXM result. It is also anticipated that a technical error of not adding patient’s serum to the donor cells can lead to positive B cell crossmatch reporting. Using m α IgG mAb or any other α IgG antibody that does not give non-specific binding to donor cells is recommended for FCXM testing.

Background In flow cytometry crossmatches (FCXM), B cell percentages of certain renal transplant patients (although not on Rituximab treatment) do not make it to laboratory set cut-off of 500 B cell events or 1000 B cell events. This is uncommon in case of donor cells. Loss of cells during washing steps is expected when cells are tagged in tubes. Aim To determine if plate method of tagging peripheral blood mononuclear cells (PBMC) will provide higher B cell percentages as compared to tube method. Methodology Peripheral blood was collected from 23 human subjects. PBMC were isolated by Ficoll density gradient separation. In 96 well V-bottom polystyrene tissue culture (TC) treated plate (Cat# 3894, Corning Inc., Corning, NY, USA), untreated plate (Cat# 651161, Greiner Bio-One, Frickenhausen, Germany), and in 5 ml polystyrene round-bottom tubes (Cat# 352054, BD, 1 Becton Drive, Franklin Lakes, NJ, USA), 300,000 PBMC were incubated with 30 μl of negative control serum (NCS) for 20 min at room temperature, washed twice, and stained with 10 μl each of anti-CD3 PerCP, anti-CD19 PE, and FITC-goat-anti-human IgG polyclonal antibody (1:100 diluted; g α IgG pAb; Jackson Immunoresearch Laboratory; Cat# 109-096-098) for 30 min at 4 °C. The cells were washed and flow cytometry was performed using BD FACSCanto II instrument and the data was analyzed using the DiVa software. Results CD19+ B cell percentages were significantly higher when the cells were tagged in TC treated plates as compared to those in tubes (9 ± 4% vs. 4 ± 2%, P = 0.0001). Expectedly, the higher B cell percentage found with cells tagged in TC treated plate were associated with a lower T cell percentages: 72 ± 13% when tagged in TC treated plates and 77 ± 12% when tagged in tubes ( P = 0.002). In addition, no difference in B cell percentages (10 ± 4 vs. 10 ± 4) or in T cell percentages (80 ± 4 vs. 80 ± 3) was identified in FCXM performed in TC treated or in untreated plates. When FCXMs were performed in tubes and in plates in parallel, channel shifts for anti-T or B cell specific IgG antibodies in human serum were identical. Conclusion This observation cautions that cells tagged or stained in plates have different percentage of cells as compared to that in tubes. In FCXM, plate method of tagging helps acquiring more B cell events as compared to tube method of tagging cells.

Aim The AbSorber flow cytometry crossmatch (FXM) XM-ONE 3:1 (XM3) test measures binding of IgG and IgM antibodies (ab) in recipient’s serum to donor-T, -B, and -Tie2+ EPC. The XM3 and our laboratory’s Clinical FXM (CFXM) were compared to examine the feasibility of the XM3’s use in clinical settings. Methods Peripheral blood (N=5) and serum (N=5) were collected from human subjects. Tie2+ EPC and lymphocytes were isolated using manufacturer’s instructions. Cells were incubated with either test serum, negative control serum (NCS) or positive control serum (PCS), washed, and stained with a combination of anti-CD3, -CD19, and ab directed at human -IgG, or -IgM. The median channel shift (MCS) from the NCS for T cell, B cell, and EPC FXM for IgG was considered positive if > 40, >100, and >50, respectively. The MCS cut-off for IgM on T cell, B cell, and EPC was considered positive if >80 MCS. The FXM were acquired in a BD FACSCanto II instrument and the data analysis was performed using the DiVa software. Results 1) The XM3 and CFXM for CD3+IgG+ T cells had similar Median Channel Fluorescence (MCF) (271 ± 19 vs. 241 ± 26) and MCS (9 ± 16 vs. 22 ± 19). All crossmatches were negative; 2) The XM3 and CFXM for CD19+IgG+ B cells also had similar MCF (301 ± 40 vs. 273 ± 19) and MCS (114 ± 77 vs. 105 ± 69). Two B cell FXM were positive and the remaining crossmatches were negative by both methods; 3) Three FXM for CD3+IgM+ T cell and CD19+IgM+ B cell by XM3 and CFXM were negative by both methods; and 4) Of five EPC crossmatches performed by XM3, two subjects were positive for IgG and all five subjects were negative for IgM. Conclusions In addition to facilitating the detection of conventional analysis of donor T and B cell specific IgG ab, the XM3 helps identify donor T and B cell specific IgM ab in recipient’s serum. In contrast to anti-HLA ab, donor-EPC specific IgG or IgM ab occur in fewer recipients, but their identification may impact allograft survival. The XM3 identifies EPC specific IgG or IgM ab in human serum.