In a prospective study of 80 patients, we investigated the association of acute kidney graft rejection with pretransplant T helper/suppressor activity, B-cell responses, and in vitro cytokine secretion. Patients' CD4+ or CD8+ T cells were cocultured with control B cells and pokeweed mitogen for 6 days. SAC I was used for T cell- and monocyte-independent B-cell stimulation and pokeweed mitogen was used for T cell-dependent B-cell stimulation. B-cell differentiation was assessed in a reverse hemolytic plaque assay. Cytokine responses of T cells (interleukin [IL]-2, IL-10, gamma-interferon) and B cells/monocytes (IL-6, IL-8, tumor necrosis factor-alpha, granulocyte-macrophage colony-stimulating factor) were determined in culture supernatants using ELISA. Subsets of CD4+ T cells, CD8+ T cells, and B cells were assessed by flow cytometry. None of 12 patients with pretransplant CD4 helper defects (CD4 helper activity < 10%) had acute rejection episodes, in contrast to 32 of 68 (47%) patients with normal pretransplant CD4 helper function (P = 0.001). Patients with pretransplant CD4 helper defects also had better 1-year graft function than patients without CD4 helper defects (serum creatinine 1.2 +/- 0.1 mg/dl and 1.7 +/- 0.1 mg/dl, respectively, P < 0.05). Pretransplant IL-10 responses were significantly associated with the occurrence of acute rejection episodes (P = 0.001) and impaired 1-year graft function (P < 0.001). All 14 patients with low pretransplant IL-10 responses (< 100 pg/ml) had 1-year serum creatinine values of < 1.5 mg/dl. Pretransplant B-cell defects and B cell/monocyte-derived cytokine secretion were not related to the incidence of graft rejection or infectious complications. Pretransplant CD8 suppressor-effector (CD11b+), cell counts were significantly associated with the occurrence of infections (P < 0.05). These results show that pretransplant CD4 helper defects and low IL-10 responses predict a low risk of graft rejection, whereas Th1 (IL-2, gamma-interferon) and B-cell/monocyte responses are not of predictive value.

Pretransplant CD4 helper function and interleukin 10 response predict risk of acute kidney graft rejection.

Selection procedures in bone marrow transplantation (BMT) would benefit from the development of easy-to-perform cellular assays with high discriminative power. We tested a cytokine-based mixed lymphocyte culture (MLC) and compared its outcome to the routinely used MLC, helper T-lymphocyte precursor (HTLp)-f and cytotoxic T-lymphocyte precursor (CTLp)-f assays. Interferon (IFN)gamma was selected as a marker cytokine for (deleterious) T-helper 1 like responses and 36 (potential) BMT donor-recipient pairs were analysed. The IFNgamma-MLC appeared sensitive to HLA class II (subtype) differences, but not to isolated class I differences, or to mismatches other than HLA (identical siblings). The test enabled a distinction between combinations with positive MLC (proliferation) and HTLp-f, exemplified by the fact that although high IFNgamma levels were observed in the class II mismatched group, certain DRB3, DQB1-subtype and DRB1-subtype mismatches did not give rise to IFNgamma production. This might be of relevance for the detection of so-called permissible mismatches. With regard to prediction of acute graft-versus-host disease (aGVHD) in unrelated BMT, the data indicated that high levels of IFNgamma coincided with severe aGVHD, whereas low levels were largely associated with grades 0-I. However, in the case of isolated class I mismatches the test had no predictive value. The cell-saving IFNgamma-MLC provides an alternative for the assays currently in use, but should be employed along with an assay that is sensitive to class I differences to correct for false negatives. Consequently, a combination of IFNgamma-MLC and CTLp-f assays seems most promising for donor selection, other than identical siblings.

Interferon-gamma-based mixed lymphocyte culture as a selection tool for allogeneic bone marrow donors other than identical siblings.

Human leucocyte antigen (HLA) compatibility is the main factor determining the occurrence of graft-vs-host disease (GVHD) in patients. It has also been shown that minor histocompatibility antigen differences as well as genetic polymorphisms that are not sequenced by standard methodology for HLA typing can play a role. We used mixed lymphocyte cultures (MLCs) as a functional cellular test and investigated gene expression changes driven by HLA incompatibility in an effort to better understand the mechanisms involved in the disease. Gene expression profile of HLA matched and HLA mismatched MLC identified differentially regulated genes and pathways. We found that a great number of genes related to immune function were differentially regulated; these genes were also found to be associated with GVHD and graft rejection. The majority of differentially regulated genes were interferon-gamma (IFNγ)-inducible genes and IFNγ neutralisation in MLCs abrogated their induction. The microRNA-155, a recently identified target for acute GVHD (aGVHD), was also found to be significantly induced in HLA mismatched MLC but not in the matched setting and its induction was not diminished by blocking IFNγ. In this proof-of-principle study we show gene expression changes in mismatched MLC that represent alloreactive responses, correlate with markers involved in GVHD and can potentially be useful in the study of the biological processes involved in this disease.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/tan.12543

Gene expression changes in HLA mismatched mixed lymphocyte cultures reveal genes associated with allorecognition.

Differential alloreactivity at SLA-DR and -DQ matching in two-way mixed lymphocyte culture.

In renal transplantation, rejection of the allograft is still a problem despite of new immunosuppressive strategies and improved HLA-matching by molecular typing methods. Survival of the graft mainly depends on the number of HLA-mismatches between donor and recipient. However, case reports revealed that even full matched cadaveric organs may undergo repeated rejection episodes resulting in the loss of the renal graft. Several mechanisms may be responsible for this poorly understood event. The alloreactivity may possibly be induced by HLA-subtypes not being characterised until now, silent HLA-mismatches or proteins of human endogenous retroviruses (HERV) which correspond to the minor lymphocyte stimulating antigens (Mis) in the mouse [1]. These Mis were first described in mixed lymphocyte culture experiments (MLC) of MHC-identical mouse strains while stimulating lymphocytes despite MHC-identity. Since perfectly HLA-matched donors were only found in very few cases [2] the problem of acceptable and non-acceptable mismatches between donor and recipient is even more obvious in transplantation medicine. Little is known on how to identify these combinations. A useful test should directly quantify the activation of T-helper-cell- (TH-) subpopulations of donor and recipient and therefore measure the biological relevance of stimulating antigens. The functional in vitro T-cell assays are used for this purpose, especially MLC. Recently, it is discussed whether measurement of cytokines in the two-way MLC may probably be used for the prediction of the rejection or the acceptance of a graft. The conventional standard oneway MLC test measures the cell proliferation between antigen-presenting cells (APC) and T-cells of host and graft and vice versa. It has been established in the clinical routine of bone marrow transplantation (BMT) for many years. However, the measurement of the proliferation in one-way MLC has no satisfying predictive value for the outcome of transplantation of either bone marrow or renal allografts [3].

Reverse transcription polymerase chain reaction for cytokines in the mixed lymphocyte culture. Is there a role in renal transplantation

Cytokine determination in MLC is under discussion as providing more sensitive and specific information regarding host-graft compatibility, and is therefore suggested to represent a new method for transplantation medicine. Little is known, however, about the stimulatory influence of HLA class II antigens and minor lymphocyte-stimulating antigens (Mls). Our results demonstrate that cytokine determination in MLC is suitable to detect identical alleles of HLA-DRB1 and HLA-DQB1. Among more than 100 random MLC experiments, we observed one cytokine pattern similar to the cytokine release detected in a control MLC of HLA-identical siblings, which showed marginal or no secretion of IL-2, sIL-2R, IFN-gamma, TNF-alpha, and IL-6. HLA-typing of these two nonreactive individuals elevated identical HLA-DRB1 and HLA-DQB1 regions, while they differed in the HLA-DP locus. This suggests that HLA-DP has no stimulatory influence on cytokine release. Further investigation of the stimulatory capacity of HLA-DR and DQ showed that HLA-DR is more effective in inducing IFN-gamma release than HLA-DQ. To evaluate the stimulatory influence of human Mls, i.e., human endogenous retroviruses (HERV), we analyzed HERV sequences of nonreactive individuals. Both individuals showed identical HERV patterns. A third individual, who had shown distinct cytokine release in MLC with both nonreactive individuals, differed in the HERV fragments. In conclusion, cytokine determination in MLC is a new method of evaluating the biological relevance of stimulatory antigens after allogeneic stimulation detecting all individual diversities in one experiment.

Identification of HLA-DRB1 and HLA-DQB1 identical individuals by a cytokine-based mixed lymphocyte culture.

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Identification of HLA-DRB1 and HLA-DQB1 identical individuals by a cytokine-based mixed lymphocyte culture.