Immune Markers Used for Diagnosis and Therapy in Connection With Transplant Reactions

Abstract

The invention relates to immune markers for detecting inflammations, immune reactions, and particularly transplant tolerance or transplant reactions, a method for detecting transplant reactions or tolerance, and the use of immune markers for medical prophylaxis, clinical monitoring of progress, transplant follow-up treatment, clinical diagnosis and/or therapy in connection with cell transplants, tissue transplants, or organ transplants.

Description

EXAMPLES

Example 1

The nucleic acid molecules according to the invention can be identified in laboratory animals, for example, in the established orthotropic kidney transplantation model of rats in which the expression of the markers according to the invention can be used for post-surgical diagnostics. In the transplantation model employed (WF donor kidneys to BDIX receptors), several applications of an anti-CD4 antibody RIB5/2 can induce tolerance towards kidney grafts which are rejected between days 5 and 9 in the control antibody of treated receptor animals. The tolerance is characterized by a long lasting normal kidney function without an increase of the serum creatinine or proteinuria for more than 300 days. The infiltration of donor-reactive T cells is reduced only to 50%, but destruction of the grafted organ does not occur.

Within the scope of the invention, the mononuclear cells immigrated into the graft were isolated from receptor animals treated with control antibodies or RIB/2 on day 5 after the transplantation by collagen digestion and Fiscoll gradient, and their mRNA expression was compared by means of the “PCR select” method. This resulted in the isolation of cDNA fragments which are expressed at an increased level in grafts of rejecting receptor animals: 2A5 and 2A15 (corresponding to SEQ ID Nos. 1 and 2).

Also, cDNA fragments could be isolated whose expression is increased in grafts of tolerance-developing receptor animals: 1A50, 3A29, T4, T5, T8 and T10 (corresponding to SEQ ID Nos. 3, 4, 5, 6, 7 and 8). FIG. 1 shows the cDNA sequence segments of the fragments mentioned.

Further, according to the invention, oligonucleotide sequences for performing a real time RT PCR were derived from the sequence segments represented here. By means of these oligonucleotide sequences, a relative quantification of the expression of the corresponding mRNAs with respect to the “house-keeping gene” β-actin is possible in rat cells. Also, by using the homologous mouse sequences, oligonucleotide sequences were established for the relative quantification of the corresponding mRNAs with respect to the “house-keeping gene” HPRT in mouse cells.

Using the thus established oligonucleotide sequences, kinetic expression studies were performed in several transplantation models within the scope of the invention. In addition to the kidney transplantation model in rats as mentioned above, the expression of the fragments was also analyzed in a heart transplantation model in mice. In this model, a donor-specific blood transfusion (B10) is administered to the receptor animals (CBA) in combination with the anti-CD4 antibody YTS177 four weeks before the transplantation. This results in the induction of a donor-specific tolerance at the time of transplantation. Control hearts in untreated receptor animals are rejected between days 7 and 8.

In FIG. 2, the results of the expression analysis are shown for the fragments 1A50, 3A29, T4, T5, T8 and T10 in the kidney transplantation model. There is shown the mRNA expression of the fragments within the graft for receptor animals treated with control antibodies (Co) on days 0 (naive kidneys), 2 and 5 after the transplantation, and in addition, there is shown the expression for RIB5/2-treated tolerance-developing receptor animals (RIB5/2) on days 0, 2, 5, 10, 14 and 300 after the transplantation. All cDNA fragments are strongly expressed in permanently accepted grafts, but in grafts of receptor animals treated with control antibodies, their expression is drastically decreased at the time of rejection.

Subsequently, the expression of the corresponding mRNAs in the heart transplantation model was examined. In FIG. 3, the expression of the fragments 1A50 and T8 in the grafted organ is shown. The mRNA expression was analyzed in grafts of pretreated tolerance-developing receptor animals (DST+YTS177) on days 0 (naive hearts), 2, 5, 7, 8, 10, 40 and 100 after the transplantation. The results were compared with the mRNA expression in the graft on untreated control animals (Co) on days 0 (naive hearts), 2, 5, 7 and 8. In the heart transplantation model, permanently accepted grafts also exhibit a high mRNA expression of 1A50 and T8. In the grafts of rejecting receptor animals, the expression is again highly reduced.

The different expression of 1A50 and T8 is also reflected in the peripheral blood. A drop of the expression of 1A50 and T8 shortly before the rejection (day 5) only occurs in the blood cells of untreated receptor animals (Co) (FIG. 4).

Further, the expression of the cDNA fragments 2A5 and 2A15 was examined in the kidney transplantation model (FIG. 5) and in the heart transplantation model (FIG. 6). The expression of these cDNA fragments in the graft of rejecting receptor animals is respectively shown. In both models, a high regulation of the mRNA expression occurs shortly before the rejection.

By means of the identification and quantification of such gene markers whose expression in the graft, in fluids from the graft (urine, lavage) or in peripheral blood correlates either with a long lasting good graft function or with the occurrence of rejections, a better evaluation of the tolerance-inducing therapy would be possible.

In the biopsy, the expression of 2A5 and 2A15 can be used for the evaluation of acute subclinical rejection crises and beginning chronic rejections. A strong and long lasting expression would be associated with the rejection of the organ. Only in a qualified way, this depends on the extent of infiltration of mononuclear cells into the graft, since the infiltration of mononuclear cells is only reduced to 50% in anti-CD4-treated tolerance-developing receptor animals in the kidney transplantation model. This would substantially improve the evaluation of a biopsy since not only infiltration into the organ is recurred to as a criterion of acute rejection, but also qualitative changes in the infiltrating cells. The expression of T4, T5, T10, 3A29, T8 and 1A50 in the biopsy can be recurred to, for example, for evaluating the success of a therapy. This would enable a decision about the safe discontinuation of the tolerance-inducing therapy.

The strong expression drop of 1A50 and T8 in the periphery in rejecting receptor animals more than 2 days before a clinical manifestation of rejection enables non-invasive diagnostics in the blood of the patient before a deterioration of the organ (e.g., increase of serum creatinine) can be detected.

The expression of 2A5 and 2A15 in the biopsy can be used for evaluating acute clinical and subclinical rejection crises and beginning chronic rejections. A strong and long lasting expression is associated with an immunological rejection of the organ. Only in a qualified way, this depends on the extent of infiltration of mononuclear cells into the graft, since the infiltration of mononuclear cells is only reduced to 50% in anti-CD4-treated tolerance-developing receptor animals in the kidney transplantation model. This substantially improves the evaluation of a biopsy since not only infiltration into the organ is recurred to as a criterion of acute rejection, but also the qualitative change of the infiltrating cells. The expression of T4, T5, T10, 3A29, T8 and 1A50 in the biopsy is recurred to for evaluating the success of the therapy. This enables a decision about the safe discontinuation of the tolerance-inducing therapy. The strong expression drop of 1A50 and T8 in the periphery in rejecting receptor animals more than 2 days before rejection enables non-invasive diagnostics in the blood or other body fluids, such as urine, of the patients before a deterioration of the organ, such as increase of serum creatinine, can be detected. Thus, the following diagnostic model after transplantation is successful:

• • 1. Detection of 1A50 and T8 in the blood or other body fluids (e.g., urine) of the patient on a daily basis shortly after the operation and on a weekly/monthly basis in the further course for predicting a rejection crisis and thus failure of the therapy, and for detecting deficient suppression in discontinuation attempts before a deterioration of the organ can be detected.
  • 2. Detection of 2A5 and 2A15 in control biopsies or graft-relevant body fluids (e.g., urine for kidney transplantation) in order to also detect rejection crises or deficient suppression in due time and to predict the risk of the development of a chronic rejection.
  • 3. Detection of T4, T5, T8, T10, 1A50 and 3A29 in control biopsies or graft-relevant body fluids in order to appreciate the success of a tolerance-inducing or conventional therapy, in particular, in order to enable the risk-free discontinuation/reduction of the therapy.

Example 2

In another animal model for tolerance, biopsies were examined from mice which accept allogenic livers spontaneously, i.e., without being influenced by drugs (liver grafts from B10 mice to CBA receptor mice), i.e., develop a spontaneous tolerance, a phenomenon which may also occur after some years subsequent to an allogenic liver transplantation. On days 0, 1, 2, 5, 7, 8, 10, 40, 100 after the transplantation, the same markers were examined in the grafts as had been previously examined upon kidney or heart transplantations. FIG. 7 summarizes the results for comparison. The spontaneous tolerance with a transient self-limiting rejection crisis in this model is reflected by a stably high expression of the tolerance markers T8 and 1A50 over the whole observation period and a but temporary increase of the rejection markers 1A6, 2A5, 2A15 in the first week after the transplantation.

This points out the clear association of the expression of the mentioned markers with tolerance or rejection in another experimental model.

Example 3

In the mouse heart transplantation model described, it could be shown that the majority of the hearts will survive on a long-term basis after tolerance induction with the protocol described, and that some hearts, however, develop signs of chronic rejection which are accompanied with a functional limitation. The latter can be determined by the “heart palpation score” (palpatory determination of the strength and rhythm of the heart beat), a high score (>3) indicating a good heart function. In a double-blind approach, the heart palpation score and expression of the tolerance markers T8 and 1A50 were determined in a comparative way and correlated with each other (FIG. 8), yielding a clear correlation between the functional ability of the heart and the expression of T8 (r=0.785) or 1A50 (r=0.784). This means that the two tolerance markers are very suitable for the prediction of incomplete tolerance, which, while not preventing acute rejection, prevents the development of a chronic rejection.

Example 4

Numerous studies show that for maintaining a stable peripheral tolerance it is necessary to form specific regulatory CD4+ T cells which apparently accumulate in the tolerant graft where they inhibit the activation and effect of effector T cells. As has been published, tolerance can be transferred to naive animals also in our models with spleen cells and even more effectively with graft-infiltrating cells (GICs) (“infectious tolerance”). In order to verify whether the mentioned tolerance markers T8 and 1A50 are overexpressed in these cells, the GICs were isolated from the grafts by means of collagenase digestion, sorted using specific antibodies and characterized in terms of their gene expression. The data show that 1A50 and even more pronouncedly T8 is highly overexpressed in GICs from grafts of tolerant animals as compared to those from rejecting animals, and that such expression becomes detectable in sorted CD4+ GICs (FIG. 9). This suggests that graft-infiltrating regulatory T cells apparently express these tolerance markers.

Example 5

The human homologues of the sequences mentioned have been identified. Now, it was tested whether the markers can also be detected by means of real-time RT PCR in biopsies and blood leukocytes of kidney-grafted patients. 1A50, 2A5 and 2A15 could be detected in all biopsies and blood samples following kidney transplantation. Patient 1 developed an acute rejection on day 23 after transplantation from a live donor. At this time, a decrease of the tolerance marker 1A50 and an increase of the rejection marker 2A15 could be observed in the peripheral leukocytes (FIG. 10). Patient 2 showed a course without complications and hardly any variations in the expression of these markers.

Further, biopsies from the grafts of kidney-grafted patients were analyzed by means of real-time RT PCR. In biopsies, patients 3 and 4 showed signs of a subclinical rejection of Banff grades Ia and Ib, respectively. The tolerance marker 1A50 exhibited a relatively low expression for rejections (especially patient 4) as compared with biopsies from patients with stable functions and without signs of rejection in the graft (patients 5 and 6) (Table 1). In contrast, the expression of the rejection marker 2A15 was highest in the two samples with rejection (patients 3 and 4) and significantly lower for a normal function (patients 5 and 6). 2A5 was similarly detectable, but showed lesser differences.

Thus, the first data from patients confirm that the genes are also detectable in a human system, and that their regulation is very similar to that observed in the animal models.

TABLE 1
Gene expression in kidney biopsies from patients with grafts
	Gene expression
	(in ratio to HPRT units)
		1A50	2A15	2A5
Patient No.	Graft function	(×10−1)	(×10−1)	(×10−1)
3	acute rejection	3.0	5.2	0.2
4	acute rejection	1.8	4.0	0.2
5	stable normal function	5.5	0.4	0.1
6	stable normal function	7.9	0.2	0.1
7	control kidney (no graft)	4.0	0.2	0.1

Claims

1-25. (canceled)

26. An isolated nucleic acid molecule selected from the group consisting of: a) a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID Nos. 1 to 8 or their complementary nucleotide sequences;b) a nucleic acid molecule which will hybridize with a nucleotide sequence according to a) under stringent conditions;c) a nucleic acid molecule comprising a nucleotide sequence which has sufficient homology with a nucleotide sequence according to a) or b) to be a functional analogue thereof;d) a nucleic acid molecule which exhibits a genetic code degeneration relationship with respect to a nucleotide sequence according to any of a) to c); ande) a nucleic acid molecule according to any nucleotide sequence of a) to d) which has been modified by deletions, additions, substitutions, translocations, inversions and/or insertions and is a functional analogue of a nucleotide sequence according to any of a) to d).

27. The nucleic acid molecule according to claim 26, wherein the nucleotide sequence as stated under c) has at least 40% homology with one of the nucleotide sequences stated under a).

28. The nucleic acid molecule according to claim 26, wherein the nucleotide sequence as stated under c) has at least 60% homology with one of the nucleotide sequences stated under a).

29. The nucleic acid molecule according to claim 26, wherein the nucleotide sequence as stated under c) has at least 70% homology with one of the nucleotide sequences stated under a).

30. The nucleic acid molecule according to claim 26, wherein the nucleotide sequence as stated under c) has at least 80% homology with one of the nucleotide sequences stated under a).

31. The nucleic acid molecule according to claim 26, wherein the nucleotide sequence as stated under c) has at least 90% homology with one of the nucleotide sequences stated under a).

32. The nucleic acid molecule according to claim 26, wherein the nucleic acid molecule is at least one of genomic DNA, cDNA or RNA.

33. A vector comprising a nucleic acid molecule according to claim 26.

34. A host cell comprising the vector according to claim 33.

35. A polypeptide encoded by a nucleic acid molecule according to claim 26.

36. A recognition molecule directed against at least one of a nucleic acid molecule according to claim 26, a vector according to claim 33, a host cell according to claim 34 or a polypeptide according to claim 35.

37. The recognition molecule according to claim 36 being at least one of an antibody, an antibody fragment or an antisense construct.

38. The recognition molecule according to claim 36 being an RNA interference molecule.

39. A vaccine comprising at least one of a nucleic acid molecule according to claim 26, a vector according to claim 33, a host cell according to claim 34, a polypeptide according to claim 35, or a recognition molecule according to claims 36 or 37 or 38, optionally with a pharmaceutically acceptable carrier.

40. A method for the detection of graft reactions in a sample from a patient, characterized in that a level of at least one nucleic acid molecule according to claim 26 is determined in the sample, and the level is compared with a control level of a comparative sample from a healthy patient, wherein the graft reactions or the absence thereof (tolerance) are detected by a modified level in the sample as compared to the control level.

41. The method according to claim 40 wherein said graft is selected from at least one of lung, spleen, heart, kidney, liver, pancreas, or tissues.

42. The method according to claim 40, wherein said graft is selected from the group consisting of islets, aortas, or cartilage.

43. The method according to claim 40, wherein a DNA or RNA concentration, gene expression, number of copies of a nucleic acid, peptide concentration, peptide activity and/or as concentration of isoforms are determined as said level.

44. The method according to claim 40, wherein said level is determined as an mRNA concentration.

45. The method according to claim 40, wherein at least one of a rejection crisis, a rejection reaction, a course of a rejection, a tolerance reaction, or a course of a tolerance is detected as said graft reaction.

46. The method according to claim 33, wherein said rejection crisis, rejection reaction or course of a rejection is detected by a reduced level of a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID No. 3 and SEQ ID No. 7 or their complementary nucleotide sequences.

47. The method according to claim 33, wherein said rejection reaction, course of a rejection or rejection crisis is detected by an increased level of a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID No. 1 and SEQ ID No. 2 or their complementary nucleotide sequences.

48. The method according to claim 40, wherein said tolerance or course of a tolerance is detected by an increased level of a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7 and SEQ ID No. 8 or their complementary nucleotide sequences.

49. Use of a nucleic acid molecule according to claim 26, vector according to claim 33, host cell according to claim 34, polypeptide according to claim 35, recognition molecule according to claims 36 or 37 or 38 and/or vaccine according to claim 39 in at least one of medical prophylaxis, clinical follow-up, graft follow-up treatment, clinical diagnostics or therapy.

50. The use of the nucleic acid molecule according to claim 49 for the detection of T-cell-mediated immune processes.

51. The use of the nucleic acid molecule according to claim 49 for the detection of pathogenic T-cell-mediated immune processes.

52. The use according to claim 50, wherein said T-cell-mediated immune processes are auto-immune diseases or inflammations.

53. The use according to claim 50, wherein said T-cell-mediated immune processes are selected from the group consisting of an antiglomerular basal membrane disease, auto-immune diseases of the nervous system, systemic lupus erythematosus, Addison's disease, antiphospholipid syndrome, IgA glomerulonephritis, Goodpasture's syndrome, Lambert-Eaton myasthenic syndrome, bullous pemphigoid, thrombocytopenic idiopathic purpura, auto-immune thyroiditis, rheumatoid arthritis, insulin-dependent diabetes mellitus, pemphigus, auto-immune hemolytic anemia, dermatitis herpetiformis Duhring, membranous glomerulonephritis, Graves' disease, sympathetic ophthalmia, auto-immune polyendocrinopathies, multiple sclerosis and Reiter's disease.

54. The use according to claim 50, wherein said T-cell-mediated immune processes are at least one of physiological, pathological, clinical or subclinical graft reactions.

55. The use according to claim 54, wherein said graft reactions include at least one of a rejection crisis, a rejection reaction, a course of a rejection, a tolerance reaction or a course of a tolerance.

56. A kit comprising at least one of a nucleic acid molecule according to claim 26, a vector according to claim 33, a host cell according to claim 34, a polypeptide according to claim 35, a recognition molecule according to claims 36 or 37 or 38, or a vaccine according to claim 39.

57. Use of the kit according to claim 56 for the detection of a graft reaction.