DE10234200A1 - Use of regulatory CD25 positive T cells for modulating inflammatory responses, for treating transplant rejection, autoimmune diseases and tumors - Google Patents

Abstract

Use of regulatory alpha-ECD25+CD4+, alpha+ECD25+CD4+ and/or alpha-ECD25-CD4+ T cells (A) of a mammal for preparing a composition for modulating inflammatory responses (alphaE = the alphaE/beta7 integrin). Independent claims are also included for the following: (1) mammalian regulatory alpha+ECD25-CD4+ T cells (A1); (2) method for identifying agents that modulate activity of (A); and (3) method for preparing a composition for modulating inflammatory responses.

Description

The present invention relates to regulatory α _E ^- CD25 ⁺ CD4 ⁺ , α _E ⁺ CD25 ⁺ CD4 ⁺ and / or α _E ⁺ CD25 ^- CD4 ⁺ T cells from mammals and their use in the manufacture of medicaments for modulating inflammatory reactions. These inflammatory reactions relate in particular to rejection after transplantation and / or an autoimmune disease, such as rheumatic inflammation, and a treatment of tumor diseases.

The so-called "immunological tolerance" is characterized by a physiological status in which the Immune system not against "body's own" Components or against harmless Antigens (e.g. food antigens) respond. The distinction between "self" and "not-self" or "danger" and "no danger" therefore plays an important role in maintaining immunological Tolerance. Immunological tolerance of T cells is at different levels developed. First it occurs when T cells mature in the thymus to a deletion of such T cells that have a high Reactivity towards self-antigens demonstrate. This step becomes "negative selection" or "central Tolerance ". There are many, but not all, self-antigens can be expressed in the thymus some T cells always leave the thymus, which have a T cell receptor express that recognizes a specific endogenous antigen which only in the periphery (somewhere in the body) and is not expressed in the thymus. Therefore there is in every organism potentially autoreactive T cells within the periphery.

• 1. Sie bilden eine Untergruppe der CD4⁺ T-Helfer-Zellen;
• 2. Der bekannteste zelluläre Marker ist CD25 (α-Kette des Interleukin-2-Rezeptors);
• 3. Sie zeigen einen "anergen" Phänotyp (niedrige Proliferationsrate und niedrige Interleukin-2-Produktion);
• 4. Sie produzieren immunsuppressiver Zytokine, wie etwa IL-10 und TGFβ;
• 5. Sie bewirken die Inhibierung der Proliferation naiver T-Zellen; und
• 6. Sie inhibieren B-Zellen und zytotoxische T-Zellen.

In order to be able to control the T cells, further levels of tolerance, the so-called "peripheral tolerance mechanisms", are required. These "peripheral tolerance mechanisms" include the regulatory T cells, which have been intensively investigated in recent years. Briefly summarized, the following characteristics of regulatory T cells are known:

• 1. They form a subset of the CD4 ⁺ T helper cells;
• 2. The best known cellular marker is CD25 (α-chain of the interleukin-2 receptor);
• 3. They show an "anergic" phenotype (low proliferation rate and low interleukin-2 production);
• 4. They produce immunosuppressive cytokines such as IL-10 and TGFβ;
• 5. They inhibit the proliferation of naive T cells; and
• 6. They inhibit B cells and cytotoxic T cells.

All of these properties above made the regulatory T cells an interesting study object, especially in the case of autoimmune diseases such as autoimmune gastritis (McHugh RS, Shevach EM, J Immunol 2002 Jun 15; 168 (12): 5979-83) or Diabetes (Gregori S, et al. Diabetes 2002 May; 51 (5): 1367-74) and inflammation, such as colitis (Singh B, Immunol Rev 2001 Aug; 182: 190-200).

Immune responses take place in either the secondary lymphoid organs (lymph nodes and spleen) or in the "affected" Tissue instead. in principle the immune response is initiated in the secondary lymphoid organs. The antigens are in the "affected" tissue by professional Antigen-presenting cells (APCs) such as dentritic cells (DCs) and processed that she presented to the T cells can be. This initial presentation does not take place in the "affected" tissue, but the antigen-laden APCs migrate the lymph in the next ("draining") lymph nodes. Here comes the contact from the Antigen-loaded APCs with the naive, i.e. hitherto antigen-inexperienced T cells, which are continuously "looking for antigen" the different secondary ones patrol lymphoid organs. When they encounter "their" antigen First, there is an increase / proliferation of the naive, antigen-specific T cells (clonal Expansion). Second, change these T cells their phenotype, since they are no longer naive and antigen-inexperienced, and take an "effector / memory" phenotype. One of those changes affects the molecules which for immigration into certain tissues is necessary (see below). Those molecules which have allowed immigration to the secondary lymphoid organs, are "switched off". Instead, the T cells now express molecules which enable them to immigrate to areas of inflammation. After clonal expansion and change in migration phenotype, leave the T cells are the secondary ones lymphoid organs and circulate through the organism until they to the area of inflammation meet in which they can now immigrate. The characterization of the migration phenotype gives information about whether the T cells tended to be secondary lymphoids Organs or in areas of inflammation can immigrate.

The immigration / migration of T cells from the blood stream / circulation into the tissue takes place through a sequence of specific interactions of the T cells with the blood vessel endothelium, which are mediated by specific molecules. This so-called "multi-step" process enables the targeted and specific immigration of T cells into the target tissue, since each of these steps must be carried out in order to enable immigration. This makes the non-specific and possibly undesirable from an immunological point of view immigration prevented.

• 1. The first step of T cell interaction with the endothelium is mediated by selectins. Are selectins molecules which bind to specific carbohydrate structures. Through this Interactions lead to a reduction in the flow rate of the T cell and due to the shear forces of blood flow to roll the T cells along the endothelium.
• 2. This slow rolling enables the T cell to move with their specific chemokine receptors bind to chemokines, which bind to the proteoglycan scaffold of the endothelial cells are immobilized. Chemokines are messenger substances that cause a hike of cells along a chemokine gradient (chemotaxis).
• 3. The binding of the chemokine to the chemokine receptor on the T cell also induces a signal which leads to the activation of specific integrins. Without this signal, the integrins are in a low affinity state. It is only through the chemokine signal that the integrins are brought into a high-affinity state, which enables firm binding to the integrin ligands on the endothelial cells. This can result in the T cells migrating into the tissue along the chemokine gradient. The three classes of molecules (selectins, chemokines and integrins) each consist of numerous members. Some of these molecules are known to have a specific function in immigration to either secondary lymphoid organs or inflammatory regions. In this context it should be mentioned that the integrin α _E β ₇ , the expression of which characterizes the investigated regulatory T cells, plays no role in the interaction with the endothelium.

In Salomon and Bluestone (Annu Rev Immunol 2001; 19: 225-52) CD28 and CTLA-4 are discussed as targets for the modulation of immune responses. These molecules play a role not only for the inflammatory reactions, but also for the inhibitory / regulatory ones. For example, CD28-mediated signals are important for the homeostasis of CD25 ⁺ CD4 ⁺ regulatory T cells and CTLA-4 is increasingly expressed on regulatory T cells. However, effects mediated by CTLA-4 are difficult to examine in detail since this molecule is both expressed on the regulatory cells and also induced on the inflammatory cells.

The US 6,383,487 describes methods for treating or preventing graft rejection based on monoclonal antibodies to CD25. Regulatory T cells are not used. From U.S. Patents 6,407,241; 6,369,204; 6,359,126; 6,358,976; 6,358,970; 6,358,915; 6,355,482; 6,352,977 inhibitors and the inhibition of integrins are also known. Regulatory T cells are also not described here.

Recently, McHugh et al. (Immunity, Vol. 16, 311-323, February 2002) described the integrin α _E β ₇ as a marker for regulatory T cells and analyzed the CD25 + CD4 + regulatory T cell population by molecular genetics. By analyzing the mRNA from these cells, it was examined which genes are expressed in the regulatory T cells. The comparison population was CD25 ^- CD4 ⁺ T cells.

It was found that, inter alia, the integrin α _E β _{7 is} increasingly expressed in the CD25 ⁺ CD4 ⁺ T cells. McHugh et al. then compared the regulatory capacity of the α _E ^- CD25 ⁺ with the α _E ⁺ CD25 ⁺ CD4 ⁺ T cells in an in vitro proliferation assay. CD25 ⁺ CD4 ⁺ and CD25 ^- CD4 ⁺ 7 cell populations were used as control cells. It turned out that the α _E ⁺ CD25 ⁺ CD4 ⁺ T cells are the most potent regulators.

Quiding-Jarbrink M et al. (Gut 2001 Oct; 49 (4): 519–25) describe the role of another beta7 integrin (α ₄ b ₇ ) in an infection model. The integrin α _E β ₇ and the use of regulatory T cells for therapy are neither described nor suggested.

WO 00/40604 describes methods and compositions for modulating the cytokine release of cells expressing alpha _E beta 7. Binding polypeptides and other molecules are mentioned which selectively bind to alpha _E beta 7 and thereby strengthen or inhibit the Th2 cytokine response. US 5,594,120 ; US 6,057,423 ; US 6,063,906 and WO 95/22610 describe the integrin alpha E subunit and antibodies directed against it, and their use.

With regard to the above Properties of regulatory T cells it is a task of present invention, specific mammalian regulatory T cells and their uses in the prevention and / or treatment of inflammatory Diseases available to provide effective therapy and against the previously known therapies for inflammatory diseases are crucial Have advantages.

This object is achieved according to a first aspect of the present invention by using a regulatory mammalian mammalian α _E ^- CD25 ⁺ CD4 ⁺ , α _E ⁺ CD25 ⁺ CD4 ⁺ and / or α _E ⁺ CD25 ^- CD4 ⁺ T cell for the manufacture of a medicament Modulation of inflammatory reactions solved.

Unless otherwise stated, the “regulatory T cells” are intended to refer to the regulatory α _E ^- CD25 ⁺ CD4 ⁺ , α _E ⁺ CD25 ⁺ CD4 ⁺ and / or α _E ⁺ CD25 ^- CD4 ⁺ T cell of a mammal can be understood. This should also be understood to mean, for example, genetically modified T cells or other cells which influence an immune reaction, as long as they can bring about the modulations of inflammatory reactions described here. The expression of the integrin α _E β _{7 characterizes} the regulatory T cells according to the invention.

As "modulation" in the sense of The present invention is a therapy when acute inflammatory reactions occur understood that both a dose of activated or inhibited regulatory T cells as well as activation or blocking of activities of regulatory T cells can include. It is said to continue below also prophylaxis of a potentially occurring inflammatory reaction and / or preventing reinforcement of the inflammatory process be understood.

The term "inflammatory response" in the sense of The present invention is to be regarded as broad and thus encompasses “classic” Inflammatory reactions, like repulsion after transplantation and / or an autoimmune reaction, especially with rheumatic illness / inflammation. Are included but also "inflammation-like" Immune reactions such as tumor diseases, especially the reaction of the immune system to tumor diseases. These are also through regulatory T cells affected and can therefore according to the present Invention can be modulated.

The use according to the invention is preferred, in which the modulation of inflammatory reactions rejection after transplant and / or an autoimmune disease, in particular rheumatic inflammation, includes.

Another aspect of the present invention relates to the use of a regulatory mammalian mammalian α _E ⁺ CD25 ^- CD4 ⁺ T cell for the manufacture of a medicament for the treatment of diseases. This subtype of regulatory T cells and its association with diseases was previously unknown. The provision of these regulatory T cells thus opens up completely new possibilities for modulating inflammatory reactions. Another aspect of the present invention thus also relates to a mammalian regulatory α _E ⁺ CD25 ^- CD4 ⁺ T cell.

• 1. Die Expression des Integrins α_Eβ₇ auf CD4+ T-Zellen aus sekundären lymphoiden Organen korreliert mit der Expression von anderen Molekülen, die als Marker für regulatorische T-Zellen gelten. Besonders sind hierbei das Molekül CD25 (siehe oben) und CD45RB hervorzuheben. Der Expressionslevel des Moleküls CD45RB unterscheidet solche T-Zellen, die noch keinen Kontakt mit Antigen hatten ("naive" T-Zellen; hohe Spiegel an CD45RB = CD45RB^high) und solche, welche schon Kontakt mit Antigen hatten ("Effektor/Memory/Gedächtnis"-Zellen; niedrige Spiegel an CD45RB = CD45RB^low). Die Mehrheit der α_Eβ₇-exprimierenden Zellen koexprimiert das Molekül CD25 (ca. 75 %) und ist in dem CD45RB^low-Kompartiment lokalisiert.
• 2. Das Integrin α_Eβ₇ unterteilt daher das bisher bekannte, "klassische" regulatorische Kompartiment (CD25⁺CD4⁺) in zwei verschiedene Zellpopulationen (α_E ⁺CD25⁺ und α_E ^–CD25⁺). Darüber hinaus konnte noch eine zusätzliche T-Zellpopulation, die nur das Integrin α_Eβ₇, aber kein CD25 exprimiert (α_E ⁺CD25^–), gefunden werden. Im Gegensatz zu der Publikation von McHugh et al. konnte erfindungsgemäß das Integrin α_Eβ₇ nicht nur als Marker für hochpotente CD25⁺CD4⁺ regulatorische T-Zellen identifiziert, sondern auch eine CD25-negative regulatorische T-Zellpopulation aufgefunden werden, welche durch das Integrin α_Eβ₇ identifziert werden kann. Dabei unterscheidet sich das Migrationsverhalten von regulatorischen T-Zellpopulationen wesentlich (siehe unten), so daß neue Möglichkeiten bei der Modulation von Entzündungsreaktionen möglich werden. Auch in der Publikation von McHugh et al. werden mögliche Differenzen zwischen den α_E ^–CD25⁺ und den α_E ⁺CD25⁺ CD4⁺ T-Zellen hinsichtlich deren Migrationsverhaltens nicht erwähnt.
• 3. Im Rahmen der vorliegenden Erfindung wurde weiterhin die regulatorische Kapazität dieser drei T-Zellpopulationen (α_E ⁺CD25^–, α_E ⁺CD25⁺ und α_E ^–CD25⁺) sowohl in einem in vitro als auch einem in vivo System untersucht. Dabei stellte sich heraus, daß die α_E ⁺CD25⁺CD4⁺ T-Zellen die potentesten regulatorischen T-Zellen sind. Aber auch die CD25-negativen Zellen (α_E ⁺CD25^–) besitzen regulatorische Aktivität.
• 4. Des weiteren konnten die Erfinder zeigen, daß die drei regulatorischen T-Zellpopulationen (α_E ⁺CD25^–, α_E ⁺CD25⁺ und α_E ^–CD25⁺) sich nicht nur hinsichtlich ihrer regulatorischen Potenz, sondern auch in Bezug auf die Expression verschiedener Zytokine und des immuninhibitorischen Moleküls CTLA-4 unterscheiden. Dieses kann als Indiz für die Verwendung unterschiedlicher regulatorischer Mechanismen angesehen werden.

The molecule α _E β ₇ belongs to the group of integrins. Integrins are heterodimers which consist of an α chain and a β chain. Integrins are expressed on different cell types and play an important role in cell-cell interaction. The integrin α _E β ₇ is known to be increasingly expressed on intraepithelial lymphocytes (IELs). In epithelial tissue, the integrin binds to epithelial cells that express the α _E β ₇ ligand - the E-cadherin. In addition, the integrin α _E β ₇ is also expressed on approximately 5-6% of the CD4 + T cells in secondary lymphoid organs (lymph nodes and spleen). In the course of the preparatory work on the present invention, these T cell populations were characterized in more detail and the following observations were made:

• 1. The expression of the integrin α _E β ₇ on CD4 + T cells from secondary lymphoid organs correlates with the expression of other molecules which are considered markers for regulatory T cells. The CD25 (see above) and CD45RB are particularly noteworthy. The expression level of the CD45RB molecule distinguishes those T cells that have not yet been in contact with antigen ("naive" T cells; high levels of CD45RB = CD45RB ^high ) and those that have already been in contact with antigen ("effector / memory / memory "Cells; low levels on CD45RB = CD45RB ^low ). The majority of the α _E β ₇ -expressing cells co-express the CD25 molecule (approx. 75%) and is located in the CD45RB ^low compartment.
• 2. The integrin α _E β ₇ therefore divides the previously known "classic" regulatory compartment (CD25 ⁺ CD4 ⁺ ) into two different cell populations (α _E ⁺ CD25 ⁺ and α _E ^- CD25 ⁺ ). In addition, an additional T cell population could be found that only expresses the integrin α _E β ₇ but no CD25 (α _E ⁺ CD25 ^- ). In contrast to the publication by McHugh et al. the integrin α _E β ₇ could not only be identified according to the invention as a marker for highly potent CD25 ⁺ CD4 ⁺ regulatory T cells, but also a CD25 negative regulatory T cell population could be found, which can be identified by the integrin α _E β ₇ . The migration behavior differs significantly from regulatory T cell populations (see below), so that new possibilities in modulating inflammatory reactions become possible. Also in the McHugh et al. possible differences between the α _E ^- CD25 ⁺ and the α _E ⁺ CD25 ⁺ CD4 ⁺ T cells with regard to their migration behavior are not mentioned.
• 3. In the context of the present invention, the regulatory capacity of these three T cell populations (α _E ⁺ CD25 ^- , α _E ⁺ CD25 ⁺ and α _E ^- CD25 ⁺ ) was further investigated in both an in vitro and an in vivo system. It turned out that the α _E ⁺ CD25 ⁺ CD4 ⁺ T cells are the most potent regulatory T cells. The CD25-negative cells (α _E ⁺ CD25 ^- ) also have regulatory activity.
• 4. Furthermore, the inventors were able to show that the three regulatory T cell populations (α _E ⁺ CD25 ^- , α _E ⁺ CD25 ⁺ and α _E ^- CD25 ⁺ ) not only differ in terms of their regulatory potency, but also in terms of expression differentiate between different cytokines and the immuno-inhibitory molecule CTLA-4. This can be seen as an indication of the use of different regulatory mechanisms.

In a few preliminary experiments, the inventors examined whether the three regulatory T cell populations (α _E ⁺ CD25 ^- , α _E ⁺ CD25 ⁺ and α _E ^- CD25 ⁺ ) also differ in the expression of certain selectins or in the responsiveness to certain chemokines ,

• 1. The α _E ^- CD25 ⁺ CD4 ⁺ T cells and the control cells (α _E ^- CD25 ^- ) show a higher frequency of L-selectin-expressing cells (CD62L ^high ) than the two α _E ⁺ CD4 ⁺ T cell populations , In this analysis, CD4 + T cells from various secondary lymphoid organs (spleen, mesenteric lymph nodes and peripheral lymph nodes) were examined. The L-selectin molecule is known to be increasingly expressed on naive, antigen-inexperienced T cells and to interact with specific carbohydrate structures that are primarily present on endothelial cells within lymph nodes. T cells that express the L-selectin in high amounts (CD62L ^high ) can therefore migrate into secondary lymphoid organs.
• 2. In contrast, the α_e ^-CD25⁺CD4⁺ T cells as well the control cells (α_e ^- CD25^-) a lower frequency of P-selectin-binding cells (P-selectin-IgG-binding) than the two α_e ⁺CD4⁺ T-cell populations. In this analysis, CD4 + T cells from various secondary lymphoids were identified Organs (spleen, mesenteric lymph nodes and peripheral lymph nodes) stained with a recombinant P-selectin-IgG fusion protein. Of the molecule P-selectin is known to be reinforced is expressed on endothelial cells which are an inflammatory region are neighboring. In contrast to L-selectin, the selectin ligand is on the T cell expressed. P-selectin is produced by inflammatory mediators like Histamine, LPS or TNFα  induced on the endothelial cells. The P-selectin ligand is only present on antigen-experienced effector / memory cells. This ensures that only Effector / memory cells in inflammatory Immigrate regions. The higher one Frequency of P-selectin binding cells within the two α_e ⁺CD4⁺ T cell populations thus indicates a preferential Migration of these cells to areas of inflammation out.
• 3. In a so-called chemotaxis experiment, the inventors tested the responsiveness of the three regulatory T cell populations (α _E ⁺ CD25 ^- , α _E ⁺ CD25 ⁺ and α _E ^- CD25 ⁺ ) to different chemokines. Total CD4 ⁺ T cells from secondary lymphoid organs were added to the upper chamber in a two-chamber system. The chemokine is in the lower chamber. The two chambers are connected to each other by a membrane that contains pores and allows cells to pass through. After certain points in time, it was analyzed which cells moved along the chemokine gradient into the lower chamber. Four different chemokines were examined. There were no differences in SDF-1, a chemokine that is important for homeostasis in non-inflammatory situations (i.e. also when migrating through secondary lymphoid organs). However, no specific chemotaxis behavior was found even with MIG, a chemokine that is formed in inflammatory regions. However, the two α _E ⁺ CD4 ⁺ T cell populations showed a lower chemotaxis compared to SLC, a chemokine that plays a central role in the migration of naive T cells into secondary lymphoid organs. However, α _E ^- CD4 ⁺ T cells were increasingly SLC responsive. In contrast, chemotaxis was increased over TARC, a chemokine that is formed in inflammatory regions, in the two α _E ⁺ CD4 ⁺ T cell populations.
• 4. In a so-called "homing" experiment, the inventors examined the tissues / lymphoid organs into which the regulatory T cell populations migrate. For this, the three T cell populations (α _E ⁺ CD25 ^- , α _E ⁺ CD25 ⁺ and α _E ^- CD25 ⁺ ) were isolated and labeled with radioactive chromium ( ⁵¹ Cr). These labeled cells were injected into the bloodstream (tail vein) of healthy mice. After three hours, the various organs were prepared and the radioactivity contained therein was measured. In this way it can be determined in which organs the respective T cell populations have migrated. It was shown that the α _E ^- CD25 ⁺ CD4 ⁺ T cells show an increased immigration into the lymph nodes (pLN, mLN, Peyer plaques), while the two α _E ⁺ CD4 ⁺ T cell populations immigrate to the liver , It is known from the liver that effector / memory cells increasingly migrate into this organ.

These experiments indicate that the two regulatory T cell populations which express the integrin α _E β ₇ (α _E ⁺ CD25 ⁺ and α _E ⁺ CD25 ^- ) and the α _E ^- CD25 ⁺ CD4 ⁺ T cells actually do distinguish their migration behavior. While the α _E ^- CD25 ⁺ CD4 ⁺ T cells have a phenotype that allows more migration to secondary lymphoid organs ("naive-like"), the two α _E ⁺ CD4 ⁺ regulatory T cell populations are more characterized by a phenotype that enables migration to inflammatory regions ("effector / memory-like").

According to the invention, the newly found "naive" α _E ^- CD25 ⁺ CD4 ⁺ cells can thus be used preventively in the modulation in "vaccinations / vaccinations" because they are likely to prevent the generation of autoreactive effector cells - that is, before the autoimmune reaction or graft rejection reaction has started. The advantage of the α _E ⁺ CD25 ⁺ CD4 ⁺ and α _E ⁺ CD25 ^- CD4 ⁺ T cells lies in their inflammatory migration behavior, as is explained in more detail in the context of the present description.

Another aspect of the present invention relates to an inventive use in combination with inflammation-modulating factors. Antibodies, in particular against integrin α _E , CD4 and / or CD25 or cytokines, in particular IL-2, IL-4, IL-5, IL-10, IL-13, TGFβ, TNFα and / or IFNγ, can be used as modulating factors , It can be used either in combination with the regulatory T cells according to the invention, or in a time-spaced manner in which the two constituents - preferably combined in a pharmaceutical packaging - are used in succession. The cytokines can also be used to further differentiate the regulatory T cells (see, for example, Barrat FJ, et al. J Exp Med 2002 Mar 4; 195 (5): 603-16; Papiernik M; Immunol Rev 2001 Aug; 182: 180-9). The use according to the invention can further include ex vivo expansion and / or pre-activation or pre-inhibition of the regulatory T cells.

Another aspect of the present invention relates to a method for finding modulatory substances of the activity of a regulatory α _E ^- CD25 ⁺ CD4 ⁺ 'α _E ⁺ CD25 ⁺ CD4 ⁺ and / or α _E ⁺ CD25 ^- CD4 ⁺ T cell of a mammal, the a) contacting a potentially modulatory substance with a regulatory α _E ^- CD25 ⁺ CD4 ⁺ 'α _E ⁺ CD25 ⁺ CD4 ⁺ and / or α _E ⁺ CD25 ^- CD4 ⁺ T cell of a mammal, and b) measuring the activity the mammalian regulatory α _E ^- CD25 ⁺ CD4 ⁺ 'α _E ⁺ CD25 ⁺ CD4 ⁺ and / or α _E ⁺ CD25 ^- CD4 ⁺ T cell. The above cells have not been used to screen for such modulators. Screening methods are known in the prior art in many different versions and these methods can be adapted to the circumstances required here without problems.

Another aspect of the present invention relates to the use of an activator of the activity of a regulatory α _E ^- CD25 ⁺ CD4 ⁺ 'α _E ⁺ CD25 ⁺ CD4 ⁺ and / or α _E ⁺ CD25 ^- CD4 ⁺ T cell of a mammal for the manufacture of a medicament for Treatment of inflammatory reactions, especially rejection after transplantation or autoimmune diseases, especially rheumatic inflammation. The activator used according to the invention induces the activity of the regulatory T cells, as a result of which their anti-inflammatory effects come to expression and the inflammation can be effectively combated.

Here, too, the activators can be administered together with administration of ex vivo expanded and / or preactivated regulatory T cells. In this case too, the migration properties of the α _E ⁺ CD4 ⁺ regulatory cells have a very positive effect.

In contrast to the activation in the case of anti-inflammation, a further aspect of the present invention relates to the use of an inhibitor of the activity of a regulatory α _E ^- CD25 ⁺ CD4 ⁺ , α _E ⁺ CD25 ⁺ CD4 ⁺ and / or α _E ⁺ CD25 ^- CD4 ⁺ T Cell of a mammal for the manufacture of a medicament for modulating immune reactions, in particular in the case of tumor diseases. In the case of tumor diseases, it is desirable that the anti-inflammatory activity of the regulatory T cells is prevented so that the tumor can be effectively combated by the body's own mechanism. Here, too, the inhibitors may be administered together with an administration of suitably modified ex vivo expanded and / or pre-inactivated regulatory T cells in order to maintain a large number of inactivated regulatory T cells in the organism and the activated regulatory T cells "stepwise adaption and dilution."

Furthermore, a method for producing a medicament for modulating inflammatory reactions is provided according to the invention, the method comprising the above-mentioned screening method. The substances identified as modulators or the derivatives (for example physiologically tolerable salts thereof) of the modulatory substance of the activity of a mammalian regulatory α _E ^- CD25 ⁺ CD4 ⁺ 'α _E ⁺ CD25 ⁺ CD4 ⁺ and / or α _E ⁺ CD25 ^- CD4 ⁺ T cells are then mixed with a pharmaceutically acceptable carrier. Methods of making such drugs are well known in the art and include, inter alia, oral, subcutaneous and / or intravenous dosage forms.

Furthermore, the invention provides a method for producing a medicament for modulating inflammatory reactions, the method comprising a) isolating regulatory α _E ^- CD25 ⁺ CD4 ⁺ , α _E ⁺ CD25 ⁺ CD4 ⁺ and / or α _E ⁺ CD25 ^- CD4 ⁺ T cells, b) the in vitro expansion of the isolated T cells, and c) optionally modulating the ex vivo isolated T cells, and d) mixing the isolated T cells with a pharmaceutically acceptable carrier. Methods for making such drugs and pharmaceutically acceptable carriers are also well known in the art and include, inter alia, oral, subcutaneous and / or intravenous dosage forms.

Also included by the present invention is a method of treating inflammatory responses in a mammal that involves the administration of mammalian regulatory α _E ^- CD25 ⁺ CD4 ⁺ , α _E ⁺ CD25 ⁺ CD4 ⁺ and / or α _E ⁺ CD25 ^- CD4 ⁺ T cells includes. Rejection after transplantation and / or an autoimmune disease, in particular rheumatic inflammation, is preferably treated as the inflammatory reaction.

According to a further aspect, the present invention relates to a method for modulating immune reactions in a mammal, the method comprising the administration of inactivated and / or the blocking of the activity of mammalian regulatory α _E ^- CD25 ⁺ CD4 ⁺ , α _E ⁺ CD25 ⁺ CD4 ⁺ and / or α _E ⁺ CD25 ^- CD4 ⁺ T cells. Preferably, a tumor disease is treated as the immune response. Furthermore, a preferred variant of the method according to the invention comprises removing the Mammalian regulatory α _E ^- CD25 ⁺ CD4 ⁺ , α _E ⁺ CD25 ⁺ CD4 ⁺ and / or α _E ⁺ CD25 ^- CD4 ⁺ T cells is performed from the patient.

In summary, the present invention results in a considerable improvement in the therapies that have been possible in the field of autoimmune diseases. The advantages of cellular therapy include minor side effects and an autologous transplant without the immunosuppression associated with significant side effects for the patient. Furthermore, a targeted modulation of the immune system is brought about, in which a simple isolation of regulatory T cells from the peripheral blood of the patients and ex vivo expansion and modulation with known cytokine cocktails is possible. Then the ex vivo expanded and possibly modulated regulatory T cells can be retransfused in the form of a specific "personalized" drug. Finally, the present invention allows the specific induction of the specific migration phenotype of the CD4 ⁺ T cells.

Since the “cellular” therapy described is very complex, since the regulatory T cell populations have to be obtained for each individual patient and expanded ex vivo in order to prevent rejection reactions against the transferred cells or so-called GVH reactions (“graft versus host”) avoid, the present invention also discloses a method for screening for modulating substances which can specifically mobilize and activate the α _E ⁺ CD4 ⁺ T cells in vivo.

The invention is now intended by following examples are further explained with reference to the accompanying drawings, but without being limited by these examples. In the figures demonstrate:

1 : Expression of α_eβ₇ Integrin on CD4⁺ T cells is with CD25 and CD45RB^low correlated.
The FACS analysis of lymphocytes from spleen and lymph nodes shows (a) the expression of α_e  and CD25 inside the CD4⁺ T cells (3.6 f 0.7% α_e ⁺CD25⁺, 9.9 + 2.5% α_e ^-CD25⁺ and 1.2 ± 0.3% α_e ⁺CD25^-; Mean ± SD of six independent Experiments), (b) expression of α_e and CD45RB on CD4⁺ T cells (a representative by four independent Experiments). The Memory (CD45RB^low) Status of α_e ⁺ Cells is not correlated with CD25 expression (data not shown).

2 : _E α ⁺ CD25 ⁺ cells do not produce pro-inflammatory cytokines after stimulation and α _E ⁺ CD25 ^- cells show a particular pattern of Th2 cytokines.
Sorted CD25 ⁺ and CD25 ^- cells were stimulated re-stimulated with PMA / ionomycin and stained for expression of CD4, α _E, CD45RB and intracellular cytokines. The frequencies of the cytokine-producing cells were analyzed by "gating" on the following subpopulations: (a) memory (CD45RB ^low ) α _E ^- CD25 ^- , (b) α _E ^- CD25 ⁺ , (c) α _E ⁺ CD25 ⁺ and (d) α _E ⁺ CD25 ^- CD4 ⁺ T cells (a representative of three independent experiments). Similar results were obtained by stimulating the cells with anti-CD3 plus anti-CD28 ( 11 ) receive. Expression of α _E and CD45RB was stable during restimulation (data not shown).

3 : α _E ⁺ CD25 ⁺ cells show the strongest regulatory capacity in vitro.
The regulatory capacities of the indicated CD4 ⁺ subpopulations were determined after 80 h co-culture with CFSE-labeled naive CD4 ⁺ T cells. As positive and negative controls, naive T cells were cultured alone with or without anti-CD3. Shown are (a) fluorescence intensities of CFSE ⁺ T cells at a regulator / target ratio of 1: 9, and (b) comparison of the regulatory capacities of all subsets at different regulator / target ratios (α _E ⁺ CD25 ⁺ / -⧠-, α _E ^- CD25 ⁺ / -Δ-, α _E ⁺ CD25 ^- / -O- or α _E ^- CD25 ^- / -⦁-). The average number of cell divisions of the CFSE ⁺ T cells is shown (a representative of six independent experiments; regulator / target ratio 1: 1 from one experiment).

4 : All regulatory T cell subsets require cell contact to perform their regulatory function.
(a) CFSE-labeled naive CD4 ⁺ T cells and the indicated CD4 ⁺ subpopulations were co-cultivated at a regulator / target ratio of 1: 3 for 80 h together with control antibodies or neutralizing antibodies against IL-10 or TGFβ. Shown is% inhibition of naive T cell proliferation (a representative of two independent experiments), (b) CFSE-labeled naive CD4 ⁺ T cells were combined with the indicated CD4 ⁺ subpopulations at a regulator / target ratio of 1: 1 for 80 h co-cultivated or the CFSE ⁺ cells and the regulatory T cells were separated by a Transwell ^™ insert. The average number of cell divisions of the CFSE ⁺ T cells is shown (a representative of two independent experiments).

5 : α _E ⁺ CD4 ⁺ T cells, CD25 ⁺ and CD25 ^- suppress induced colitis in SCID mice.
(a) SCID mice received 3 x 10 ⁵ CD45RB ^high CD4 ⁺ T cells alone (-∎-, n = 17) or in combination with 1 × 10 ⁵ CD4 ⁺ T cells from each subpopulation (regulator / target ratio 1: 3 ): CD45RB ^low (-⦁-, n = 4), α _E ⁺ CD25 ⁺ (-⧠-, n = 4), α _E ^- CD25 ⁺ (-Δ-, n = 4) or α _E ⁺ CD25 ^- ( -O-, n = 4). The progression of colitis is tracked by the adjusted body weight loss. (b) SCID mice received 3 × 10 ⁵ CD45RB ^high CD4 ⁺ T cells alone (-∎-, n = 7) or in combination with 5 × 10 ⁴ CD4 ⁺ T cells from each subpopulation (regulator / target ratio 1: 6): α _E ⁺ CD25 ⁺ (-⧠-, n = 8), α _E ^- CD25 ⁺ (-Δ-, n = 8) or α _E ⁺ CD25 ^- (-O-, n = 8). Control mice that received only PBS alone (-⦁-, n = 5) do not differ from mice that received CD45RB ^high CD4 ⁺ T cells plus CD45RB ^low in a ratio of 3: 1 (data not shown).

Fig. 6 : Intestinal pathology in SCID mice with CD45RB ^high CD4 ⁺ T cells and subsets from re gulatory CD4 ⁺ T cells were reconstituted. SCID mice received 3 × 10 ⁵ CD45RB ^high CD4 ⁺ T cells alone or in combination with 1 × 10 ⁵ CD4 ⁺ T cells from each subpopulation (regulator / target ratio 1: 3). Tissues were removed for histological examination after the examination period. Representative photomicrographs show severe colitis (transmural infiltration with ulceration) in mice that received CD45RB ^high CD4 ⁺ T cells alone (a) and complete protection of colitis in mice that additionally received CD45RB ^low (b), α _E ⁺ CD25 ⁺ (c ), α _E ^- CD25 ⁺ (d) or α _E ⁺ CD25 ^- (e) CD4 ⁺ T cells was given.

7 : FACS re-analysis of sorted CD4 ⁺ T-cell subsets.
The cells obtained from the sorting process described in Materials and Methods were reanalysed using flow cytometry. Data from a representative from six experiments are shown.

8th : Expression of CTLA-4 correlates with α _E and only to a lesser extent with CD25. Cells isolated from lymphoid tissues were stained for the surface expression of CD4, α _E and CD25 as well as for the intracellular expression of CTLA-4. Shown is CTLA-4 expression (bold line) plus isotype control (dotted line) within the given CD4 ⁺ subpopulations (numbers indicate the frequency of CTLA ⁺ cells; a representative of five independent experiments).

9 : Stability of α _E expression on CD4 ⁺ T cell subsets after in vitro stimulation.
The expression of α _E (a) and CD25 (b) on the indicated CD4 ⁺ T cell subsets was determined after 80 h stimulation within the in vitro proliferation test (numbers indicate the frequency of positive cells). One of six (α _E ) or two (CD25) experiments is shown.

10 : Suppression by the regulatory T cell subsets is not mediated by killing mechanisms.
(a) Assorted CD25 ⁺ and CD25 ^- cells isolated from combined lymphoid tissues were re-stimulated with APC plus anti-CD3 (1 µg / ml) for 18 h and then for expression. stained by CD4, α _E and Fast. Shown is the fast expression (bold line) plus isotype control (dotted line) within the specified CD4 ⁺ subpopulations. Expression of α _E was stable during restimulation (data not shown), (b) The regulatory capacities of the CD4 ⁺ subpopulations indicated, isolated from lymphoid tissues from perforin deficient or C57B16 mice, were co-cultured with CFSE-labeled naive CD4 ⁺ after 80 h T cells from C57B16 mice (regulator / target ratio 1: 3) were determined. The average number of cell divisions of the cells is shown (a representative of two independent experiments).

11 : Similar cytokine expression patterns after restimulation with anti-CD3 plus anti-CD28.
Sorted CD25 ⁺ and CD25 ^- cells were bound to plates anti-CD3 plus anti-CD28 (5 ug / ml each) restimulated and stained for expression of CD4, α _E CD45RB and intracellular cytokines. The frequencies of cytokine-producing cells were analyzed by "gating" on the following subpopulations: memory (CD45RB ^low ) α _E ^- CD25 ^- , α _E ^- CD25 ⁺ , α _E ⁺ CD25 ⁺ and α _E ⁺ CD25 ^- CD4 ⁺ T- Cells Expression of α _E and CD45RB was stable during restimulation (data not shown).

Tabelle 1: α_E ⁺CD25⁺CD4⁺ T-Zellen zeigen das höchste regulatorische Potential in vivo.

Table 1: α _E ⁺ CD25 ⁺ CD4 ⁺ T cells show the highest regulatory potential in vivo.

SCID mice received 3 × 10 ⁵ CD45RB ^high CD4 ⁺ T cells alone or in combination with CD4 ⁺ T cell subpopulations as indicated, control mice received only PBS. The clinical and histological values are shown as mean values. The occurrence of colitis was determined by the overall analysis of body weights plus clinical and histological parameters. The data for mice that received either CD45RB ^high or CD45RB ^high plus CD45RB ^low CD4 ⁺ T cells are composed of the two independent experiments.

Materials and methods

mice

Female Balb / c and C57B16 mice were found kept in our animal stable and at the age of 6-12 weeks used. For analysis of cytokine expression using Balb / c mice approximately 10 months old. Perforin-deficient mice (Kagi, D., et al. (1994) Nature 369, 31-7) have been kind by U. Steinhoff posed. Female C.B-17 SCID were from Charles River (Sulzfeld, FRG) obtained and used at the age of 5-9 weeks. All animal experiments were made under specific pathogen-free conditions and in accordance with institute, state and federal guidelines.

Antibodies, staining and sorting reagents

The following antibodies were purified and labeled in our laboratory: anti-FcR II / III (2.4G2), anti-CD3 (145.C11), FITC- and Cy5-labeled anti-CD4 (GK1.5), biotinylated anti-α _E (M290), anti-CTLA-4 (4F10), anti-IL-10 (JES5.2A5.7G4), PE-labeled anti-IL-13 (382.13.11) and rat IgG ₁ isotype control (1A5). The following antibodies and secondary reagents (FITC-, PE-, Cy5-, APC-labeled or biotinylated) were purchased from BD Phar-Mingen (Heidelberg, FRG): anti-CD4 (RM4-5), anti-CD62L (Mel- 14), anti-CD25 (PC61), anti-CD25 (7D4), anti-CD38 (90), anti-CD45RB (16A), anti-CD44 (IM7), anti γδTCR (GL3), anti-FasL (MLF3) , anti-IL-2 (JES6-5H4), anti-IL-4 (11B11), anti-IL-5 (TRFK5), anti-IL-10 (JESS-16E3), anti-TNFα (MP6-XT22), anti-IFNγ (XMG1.2), streptavidin (SA), anti-hamster IgG, and isotype controls. Polyclonal anti-TGFβ was purchased from R&D (Wiesbaden, FRG). All microbeads were obtained from Miltenyi Biotec (Bergisch-Gladbach, FRG).

Cell cleaning and culture conditions

CD4 ⁺ T cell subsets were isolated from pooled spleen, peripheral and mesenteric lymph nodes (pLN, mLN) from Balb / c mice. CD4 ⁺ T cells were enriched using the MACS MultiSort kit and the AutoMACS magnetic separation system (Miltenyi Biotec). The different regulatory subpopulations were separated as follows: after staining CD4 ⁺ T cells with biotinylated anti-α _E , PE-labeled SA and anti-PE MicroBeads, the α _E ⁺ cells were isolated using AutoMACS technology (Miltenyi Biotec). Thereafter, these α _E -enriched cells with APC-labeled anti-CD25 were stained, and the α _E α ⁺ CD25 ⁺ and _e ⁺ CD25 ^- subsets were separated by FACS Vantage ^TM sorter (BD, Heidelberg, FRG). α _E ^- CD4 ⁺ were stained with biotinylated anti-CD25 and anti-Biotin MicroBeads and the α _E ^- CD25 ⁺ as well as the α _E ^- CD25 ^- subsets were isolated using the AutoMACS. Naive CD4 ⁺ CD62L ⁺ cells were positive by _E α ^- selected fraction on a AutoMACS by the use of anti-CD62L microbeads ^- CD25. The antigen presenting cells (APC) were prepared by depleting CD90 ⁺ cells from Balb / c spleen cells using anti-CD90 microbeads and irradiated (30 Gy) before culture. CD45RB ^high and CD45RB ^low CD4 ⁺ 7 cells were sorted by FACS. All sorted subsets were> 95–99% pure after re-analysis (data for the examined subsets. See 7 ). The cell cultures were carried out with RPMI1640 (Gibco BRL, Eggenstein, FRG) plus 10% FCS (Linaris, Wertheim-Bettingen, FRG).

flow cytometry

Flow cytometric analysis was performed as previously described (Assenmacher, M., et al. (1994) Eur J Immunol 24, 1097-1101) using FACS Calibur ^™ (BD Biosciences) and CellQuest ^™ software. For CTLA-4 detection, the cells were stained for the surface expressions of CD4, α _E and CD25, fixed and permeabilized. The intracellular expression of CTLA-4 was demonstrated with hamster anti-mouse CTLA-4 and Cy5-labeled anti-hamster IgG. To analyze the expression of cytokines of particular subpopulations were MACS sorted CD25 ⁺ and CD25 ^- cells for 4 hours with PMA (10 ng / ml) plus ionomycin (250 ng / ml) or, alternatively, for 5 hours with plate-bound anti-CD3 plus anti CD28 (5 μg / ml each) stimulated with the addition of Brefeldin A (10 μg / ml) after 2 hours. Before fixation and intracellular staining were CD25 ⁺ and CD25 ^- cells on surface expression of CD4 and α _E stained; were also stained cells with anti-CD45RB ^- while CD25. PMA / ionomycin stimulation did not change the expression of α _E and CD45RB (data not shown). The non-specific binding was blocked by the addition of whole rat IgG (Jackson ImmunoResearch, Dianova, Hamburg, FRG).

In vitro cell division test

Unlabeled CD4 ⁺ T cell subpopulations were mixed with CSFE-labeled naive T cells in various regulator / target ratios (5-carboxyfluoresceindiacetate succinimidyl ester; Molecular Probes, Leiden, NL; staining as previously described, Lyons, AB & Parish, CR (1994) J Immunol Methods 171, 131-7) and with APC in a ratio of 1: 2 in the round bottom microtiter plates without addition of anti CD3 (1 μg / ml) for 80 h in 2-fold batches. Blocking antibodies were used at final concentrations of 20 μg / ml. The Transwell chambers (0.4 µm pore size) were purchased from Costar (Cambridge, MA, USA). After the incubation, the cells were collected, stained for CD4 and analyzed by FACS; Propidium iodide was added to exclude dead cells. The proliferation analysis is based on CFSE ⁺ CD4 ⁺ T cells. The average number of cell divisions (d) was calculated using the formula: d = Σ (n _i / n _t xg _i ), where g; = Generation number, starting from 0 for undivided cells, n _i = number of cells within each generation and n _t = total cell number.

T cell reconstitution

CB-17 SCID mice were injected ip with sorted CD4 ⁺ T cell subsets in PBS. The mice received CD45RB ^high CD4 ⁺ T cells alone or in combination with CD4 ⁺ T cell subpopulations (CD45RB ^low , α _E ⁺ CD25 ⁺ , α _E ^- CD25 ⁺ or α _E ⁺ CD25 ^- ) at ratios and cell numbers as indicated.

Clinical and histological investigation

Weight changes in reconstituted CB-17 SCID mice were evaluated weekly and monitored with clinical symptoms (0: no sign of clinical progression; 1: curved abdomen, impaired movement or shaggy fur; 2: liquid stool; 4: rectal prolapse). For histology, intestinal tissue samples were taken approximately 10 weeks after T cell reconstitution and in 10% phosphate-ge buffered formalin fixed. Paraffin-embedded sections were stained with hematoxylin and eosin. The degree of inflammation of the intestine was rated "in a blind way" semi-quantitatively from 0 to 4 (0: no signs of inflammation; 1: very low levels of and little focal infiltrating mononuclear cells; 2: low levels and only focal infiltration of mononuclear cells; 3: high levels of mononuclear cell infiltration, high vascular density, thickening of the intestinal wall; 4: transmural inflammatory infiltration, loss of calyx cells, high vascular density, vasculitis, thickening of the intestinal wall). The severity of colitis was determined by both values along with body weight changes A total score ≥ 3 was considered moderate or severe colitis All animals showed no inflammatory signs in the small intestine (data not shown).

statistics

The data were given as mean ± standard deviation (SD). The significance was confirmed by the Student's t test (CTLA-4), the Wilcoxon test (cell division test), repeated measurement analyzes (Body weight) and the Mann-Whitney test (severity of colitis). The differences were considered statistically significant with p ≤ 0.05 and highly significant with p ≤ 0.01.

Results:

The α _E β ₇ expression on CD4 ⁺ T cells in lymphoid tissues is correlated with CD25 and CD45RB ^low expressions.

The presence of a small population of α _E expressing CD4 ⁺ T cells has been known for a long time, but their functional role has remained unclear. The inventors therefore characterized α _E ⁺ CD4 ⁺ T cells from combined lymphoid tissues, which comprise between 2 and 6% of the total CD4 ⁺ cells in spleen, pLN and mLN and are almost exclusively αβTCR ⁺ cells (>98%; data not shown ). Flow cytometry analysis showed that approximately 75% of α _E expressing CD4 ⁺ T cells are CD25 ⁺ ( 1a ). Furthermore, α _E + CD4 ⁺ T cells were mainly found in the memory (CD45RB ^low ) compartment ( 1b ) found regardless of their CD25 expression and expressed CD38 and high levels of CD44 (data not shown). Consistent with its inhibitory function, CTLA-4 was found on CD4 ⁺ regulatory cells in previous work and was suspected to be involved in its function (Read, S., et al. (2000) J Exp Med 192, 295-302 .; Salomon, B., et al. (2000) Immunity 12, 431-40; Takahashi, T., et al. (2000) J Exp Med 192, 303-10). The inventors therefore examined the coexpressions of CTLA-4 with α _E and CD25 on CD4 ⁺ T cells ( 8th ). Approximately 18% of cells within the α _E α ⁺ CD25 ⁺ and the CD25 ⁺ _E ^- fraction of CD4 ⁺ T cells expressed high levels of intracellular CTLA-4 (18.8 ± 5.7% and 17.6 ± 5, 7%), whereas only 6.8 ± 3.2% and 0.5 ± 0.3% of that in the α _e ^- CD25 ⁺ or α _e ^- CD25 ^- subpopulation did (p <0.01, n = 5 ). Therefore, the expression of CTLA-4 correlates with that of α _E β ₇ integrin on CD4 ⁺ T cells.

Different cytokine profiles from α _E ⁺ subsets.

Regulatory CD25 ⁺ CD4 ⁺ T cells have previously been reported to produce low levels of pro-inflammatory cytokines compared to other antigen-experienced CD4 ⁺ T cells (Thornton, AM & Shevach, EM (1998) J Exp Med 188, 287- 96; Takahashi, T., et al. (1998) Int Immunol 10, 1969-80; Asano, M., et al. (1996) J Exp Med 184, 387-96; Papiernik, M. & Banz, A. (2001) Microbes Infect 3, 937-45). Within the α _E ^- CD25 ⁺ fraction, the frequencies of TNFα, IFNγ or IL-2 producing cells after restimulation were lower compared to the frequencies under CD45RB ^low memory cells. Th2 cytokines such as IL-4, IL-5 or IL-13 were less reduced or even slightly higher ( 2 ). In contrast, the α _E ⁺ CD25 ⁺ population showed almost no IL-4, IL-5 and IL-13 and very low numbers of cells produced TNFα, IFNγ or IL-2. The only cytokine still present among α _E ⁺ CD25 ⁺ cells was IL-10.

The α _E ⁺ CD25 ^- cells show a unique cytokine expression patterns.

While the frequencies of IL-2 and IFNγ positive cells were almost the same as in Memory CD4 ⁺ T cells, the frequencies for TNFα and IL-10 were lower, slightly increased for IL-10 and much higher for IL-5 and IL-13 ( 2 ). The various Zytokinproduzenten under the α _E ⁺ CD25 ^- subset showed similar levels of α _E expression.

α _E expression characterizes the most potent regulatory CD25 ⁺ CD4 ⁺ T cells.

The regulatory capacity of CD4 ⁺ T cell subsets, characterized by α _E and CD25 express analysis, the inventors separated four populations (α _E ⁺ CD25 ⁺ , α _E ^- CD25 ⁺ , α _E ⁺ CD25 ^- and α _E ^- CD25 ^- , see 7 ) and investigated their suppressive activity in the in vitro proliferation test. Naive CD4 ⁺ T cells were labeled with CFSE and stimulated with anti-CD3 and APC for 80h. During this time, the majority of naive T cells had divided several times, which was indicated by the loss of CFSE content (positive control; 3a ). The addition of α _E ⁺ CD25 ⁺ cells at a regulator / target ratio of 1: 9 showed almost complete inhibition of naive T cell proliferation, whereas α _E ^- CD25 ⁺ cells only inhibited proliferation to a lesser extent. Remarkably, even designated α _E ⁺ CD25 ^- cells to a significant regulatory capacity, leads to a significant inhibition of the naive T-cell proliferation. α _E ^- CD25 ^- cells were used as a control population without any suppressive activity.

The relative capabilities of the subsets were examined more closely by titrating the regulator / target ratios ( 3b ). At a 1:19 ratio, only α _E ⁺ CD25 ⁺ showed a significant inhibitory effect on naive T cell proliferation. Both α _E ^- CD25 ⁺ and α _E ⁺ CD25 ^- cells showed inhibitory effects at higher ratios (1: 9 and 1: 3) but remained behind the α _E ⁺ CD25 ⁺ population back (p <0.005). At a ratio of 1: 1, the proliferation of naive T cells was completely inhibited by all 3 subsets and CD45RB ^low cells ( 3b and data not shown). The subsets expressing α _E remained positive during the culture period, whereas in the subset of α _E ^- CD25 ⁺ a fraction of cells appeared within 80 hours which express low levels of the α _E. In contrast, CD25 was present on all T cells, regulatory subsets and naive target cells after 80 hours of stimulation ( 9 ). Therefore, α _E + CD4 ⁺ cells have T-lymphoid tissues of regulatory capacity and α _E ⁺ CD25 ^- subset provides a novel regulatory CD4 ⁺ T-cell population is outside of the CD25 ⁺ compartment.

To investigate whether the inhibitory effect of the regulatory subset is mediated by soluble immunosuppressive cytokines, IL-10 and TGFβ were blocked in the in vitro proliferation tests. The addition of neutralizing antibodies against IL-10 or TGFß had no strong effect on the inhibition of naive T cell proliferation by all regulatory subsets. Only the α _E ⁺ CD25 ^- cells showed a slightly reduced inhibitory capacity when TGF was blocked ( 4a ). In addition, it was found that the inhibitory function of all regulatory subsets is dependent on cell contact, since no suppression of naive T cell proliferation was observed when the regulatory cells and the target cells were cultured through Transwell chambers ( 4b ) were separated.

α _E + CD4 ⁺ T cells inhibit the development of induced SCID colitis.

In order to analyze the function and the potential of the examined CD4 ⁺ T cell subpopulations in vivo, their ability to develop colitis after transfer of naive (CD45RB ^high ) CD4 ⁺ T cells in SCID mice was tested. In a first series of experiments, mice reconstituted with CD45RB ^high CD4 ⁺ T cells alone developed colitis (n = 17) in 88% of cases, whereas none with naive cells plus either α _E ⁺ CD25 ⁺ , α _E ^- CD25 ⁺ , α _E ⁺ CD25 ^- or CD45RB ^low CD4 ⁺ T cells (3: 1 ratio) reconstituted mice developed colitis. The body weights of all mice receiving protective CD4 ⁺ T cells (α _E ⁺ CD25 ⁺ , α _E ^- CD25 ⁺ , α _E ⁺ CD25 ^- or CD45RB ^low ) in addition to naive cells increased to a similar extent, whereas that of mice reconstituted with naive cells alone (p = 0.005, 5a ). Histological examination of colon tissue samples from mice reconstituted with naive cells alone showed transmural and in some cases only focal mononuclear infiltrates and the loss of calyx cells ( 6a ). In contrast, colonies that were restored with a mixture of naive cells plus either α _E ⁺ CD25 ⁺ , α _E ^- CD25 ⁺ , α _E ⁺ CD25 ^- showed no detectable pathological changes ( 6b-e ). These results show that α _E ⁺ subsets are effective regulators of intestinal inflammation in vivo both inside and outside the CD25 ⁺ CD4 ⁺ compartment.

In order to determine whether the investigated regulatory populations also differ in their suppressive capacity in vivo, as was found in vitro, SCID mice were reconstituted in a second experimental series using lower regulator / target ratios. Analysis of the body weights of mice reconstituted with naive plus protective CD4 ⁺ T cells at a ratio of 6: 1 showed that α _E ⁺ CD25 ⁺ cells also had the highest regulatory potential in vivo ( 5b ). Only one mouse in this group (n = 8) developed colitis. In contrast, reconstructions with α _E ⁺ CD25 ^- or α _E ^- CD25 ⁺ cells, in addition to naive CD4 ⁺ T cells, only protected 50% of the mice (n = 8) from the onset of colitis (Table 1). The comparison of the body weights of mice that received either α _E ⁺ CD25 ⁺ or α _E ^- CD25 ⁺ cells in combination with naive CD4 ⁺ T cells showed an almost significant difference (p = 0.056), which was shown by the entire statistical analysis of Body weight plus clinical and histological parameters was confirmed (Table 1). These data clearly show whether the α _E + CD25 ⁺ also has the highest regulatory capacity among the subsets described so far in vivo. Interestingly, the α _E ⁺ CD25 showed ^- Subset a remarkable protective effect in vivo, in spite of its relatively low efficiency in the in vitro tests.

Claims (17)

Use of a regulatory mammalian mammalian α _E ^- CD25 ⁺ CD4 ⁺ , α _E ⁺ CD25 ⁺ CD4 ⁺ and / or α _E ⁺ CD25-CD4 ⁺ T cell for the manufacture of a medicament for modulating inflammatory reactions. Use according to claim 1, characterized in that the modulation of inflammatory reactions the repulsion after transplant and / or an autoimmune disease, in particular rheumatic inflammation, and / or tumor disease. Regulatory α _E ⁺ CD25 ^- CD4 ⁺ T cell of a mammal for the treatment of diseases. Use according to one of claims 1 to 3 in combination with inflammatory modulators Factors. Use according to claim 4, characterized in that the inflammation- _modulating factors are antibodies, in particular against integrin α _Έ , CD4 and / or CD25 or cytokines, in particular IL-2, IL-4, IL-5, IL-10; IL-13, TGFß, TNF? and / or IFN? be used. Use according to any one of claims 1 to 5, further comprising an ex vivo expansion and / or pre-activation or pre-inactivation of regulatory T cells. Mammalian regulatory α _E ⁺ CD25 ^- CD4 ⁺ T cell. A method for finding modulatory substances of the activity of a regulatory α _E ^- CD25 ⁺ CD4 ⁺ , α _E ⁺ CD25 ⁺ CD4 ⁺ and / or α _E ⁺ CD25 ^- CD4 ⁺ T cell of a mammal, comprising a) contacting a potentially modulatory Substance with a regulatory α _E ^- CD25 ⁺ CD4 ⁺ , α _E ⁺ CD25 ⁺ CD4 ⁺ and / or α _E ⁺ CD25 ^- CD4 ⁺ T cell of a mammal, and b) measuring the activity of the mammalian regulatory α _Έ ^- CD25 ⁺ CD4 ⁺ , α _E ⁺ CD25 ⁺ CD4 ⁺ and / or α _E ⁺ CD25 ^- CD4 ⁺ T cell. Use of an activator of the activity of a regulatory α _E ^- CD25 ⁺ CD4 ⁺ , α _E ⁺ CD25 ⁺ CD4 ⁺ and / or α _E ⁺ CD25 ^- CD4 ⁺ T cell of a mammal for the manufacture of a medicament for the treatment of inflammatory reactions, in particular rejection after Transplant or autoimmune diseases. Use of an inhibitor of the activity of a regulatory α _E ^- CD25 ⁺ CD4 ⁺ , α _E ⁺ CD25 ⁺ CD4 ⁺ and / or α _E ⁺ CD25 ^- CD4 ⁺ T cell of a mammal for the manufacture of a medicament for the treatment of inflammatory reactions, in particular tuμoral diseases. A method for producing a medicament for modulating inflammatory reactions, comprising the method according to claim 8 and mixing the modulatory substance with the activity of a mammalian regulatory α _E ^- CD25 ⁺ CD4 ⁺ , α _E ⁺ CD25 ⁺ CD4 ⁺ and / or α _E ⁺ CD25 ^- CD4 ⁺ T cell with a pharmaceutically acceptable carrier. Process for the manufacture of a medicament for modulation of inflammatory reactions, full - Isolation of regulatory α_e ^-CD25⁺CD4⁺, α_e ⁺CD25⁺CD4⁺ and / or α_e ⁺CD25^- CD4⁺ T cells - in vitro expansion the isolated T cells, - possibly Activate or inhibit the isolated T cells, and - Mix of the isolated T cells with a pharmaceutically acceptable carrier. A method of treating inflammatory responses in a mammal, comprising administering mammalian regulatory α _E ^- CD25 ⁺ CD4 ⁺ , α _E ⁺ CD25 ⁺ CD4 ⁺ and / or α _E ⁺ CD25 ^- CD4 ⁺ T cells. A method according to claim 13, characterized in that the inflammatory reaction a rejection after Transplant and / or an autoimmune disease, in particular rheumatic inflammation, includes. A method for modulating immune responses in a mammal comprising administering inactivated and / or blocking the activity of mammalian regulatory α _E ^- CD25 ⁺ CD4 ⁺ , α _E ⁺ CD25 ⁺ CD4 ⁺ and / or α _E ⁺ CD25 ^- CD4 ⁺ T- cells. A method according to claim 15, characterized in that the immune response includes a tumor disease. The method of claim 15 or 16, characterized in that the blocking removal of the mammalian regulatory α _E ^- CD25 ⁺ CD4 ⁺ ; α _E ⁺ CD25 ⁺ CD4 ⁺ and / or α _E ⁺ CD25 ^- CD4 ⁺ T cells from the patient.