Article

ISSN 0006-2979, Biochemistry (Moscow), 2025, Vol. 90, No. 2, pp. 161-172 © Pleiades Publishing, Ltd., 2025.

Russian Text © The Author(s), 2025, published in Biokhimiya, 2025, Vol. 90, No. 2, pp. 175-188.

161

REVIEW

Memory T Cells: Investigation of Original Models

with Transgenic T Cell Receptors

Dmitry B. Kazansky

1,a

*, Anastasiia A. Kalinina

, and Ludmila M. Khromykh

N. N. Blokhin National Medical Research Center of Oncology, Ministry of Health of the Russian Federation,

115522 Moscow, Russia

e-mail: kazansky1@yandex.ru

Received October 31, 2024

Revised December 26, 2024

Accepted December 27, 2024

Abstract— This review summarizes the research data on original mouse models developed in the laboratory

of regulatory mechanisms in immunity of the Research Institute of Carcinogenesis, N.  N.  Blokhin National

Medical Research Center of Oncology, Ministry of Health of the Russian Federation. Transfer of the genes of

individual α- and β-chains of T cell receptors (TCRs) of memory cells has resulted in production of transgenic

animal lines valuable for studying T lymphocyte homeostasis and patterns of formation of their activation

profile markers. Investigation of the transgenic models revealed new features of immune selection and tu-

mor progression. In particular, the fundamental property of some TCRs, termed “chain-centricity”, has been

confirmed; it involves dominance of one of the TCR chains during recognition of the MHC (major histocom-

patibility complex)/peptide complex. This property makes it possible to artificially generate a significant pool

of immunocompetent T  cells so it could be used in adoptive immunotherapy for oncological and infectious

diseases. Transfer of the dominant active TCR α-chains provides the possibility for constructing organisms

with innate specific immunological resistance to certain pathogens. The results of recent studies indicate

that TCR, determining the T lymphocyte relationship with its MHC microenvironment, has an instructive

role in formation of its functions and phenotype. One of these functions may be production of cyclophilin

A by the cortisone-resistant memory cells localized in thymus. The evidence has been accumulated that

expression of TCR with a certain structure and specificity is a sufficient condition for formation of the func-

tional potential of memory cells in a T  cell, regardless of its former interaction with antigenic MHC/peptide

complexes.

DOI: 10.1134/S0006297924603940

Keywords: T lymphocyte, repertoire, major histocompatibility complex, peptide, thymus, T cell receptor,

chain-centricity, cyclophilin A, transgenesis, adaptive immunity, adoptive immunotherapy

Abbreviations: MHC, major histocompatibility complex; TCRs, T cell receptors.

* To whom correspondence should be addressed.

INTRODUCTION

In the middle of the last century, studies of spe-

cific immunity and immunological tolerance led im-

munologists to realize discrete nature of the carri-

ers performing functions of adaptive immunity. This

understanding resulted in suggestion of the clonal

selection theory of immunity. According to this the-

ory, adaptive immunity is performed by lymphocyte

clones, each of which produces receptors with unique

specificity. Within this theory, immunological toler-

ance could easily be explained by the death or inac-

tivation of the lymphocyte clones expressing receptors

for a tolerant antigen, while specific immunity – by

activation and multiplication of the clones carrying

receptors for an immunogenic antigen. The ability of

some of these clones to persist for a long time, in turn,

explained the phenomenon of immunological memory,

which is the basis of vaccination methods protecting

an organism against dangerous infections. Develop-

ment of the T and B lymphocyte cloning techniques

and further research of the structure and mechanisms

KAZANSKY et al.162

BIOCHEMISTRY (Moscow) Vol. 90 No. 2 2025

of formation of the unique lymphocyte receptors have

proved beyond any doubts validity of the clonal selec-

tion theory of immunity and its scientific and prog-

nostic significance and effectiveness. Further develop-

ment of the recombinant DNA technologies, as well as

transfection, transduction, and transgenesis methods

opened up a possibility of a wide scope of work in

the field of genetic modification of lymphocytes and

organisms using adaptive immunity receptor genes

and investigation of acquired new properties and

mechanisms of the immune system homeostasis. Full

implementation of this approach in studying immu-

nological memory was impossible due to involvement

of naive T

  lymphocytes in the secondary immune re-

sponse. Their activation by an antigen leads to acqui-

sition of an activation marker profile similar to that

of the memory cells, which makes it impossible to dif-

ferentiate them and unambiguously attribute certain

properties to the specific cell type [1]. This situation

was further complicated by acquisition of the memo-

ry cell markers by T

  lymphocytes that occurred under

lymphopenia in the absence of antigenic stimulation,

which made the subject of research such as mem-

ory T

  cells even more uncertain [2]. The term “vir-

tual memory cells” have appeared in the literature,

but their real relationship to immunological memory

remains unclear, along with the growing conviction

that the T

cells proliferating under lymphopenia are

autoreactive [3,  4]. At the same time, interpretation

of the results of studies focused on the memory cells

obtained from the models constructed with transgenic

T  cell receptors (TCRs) did not provide any support

to the notion that the investigated transgenic TCRs

originated from the long-lived memory cells subjected

to prolonged selection during the immune response

invivo. In this context, the T  lymphocytes with a trans-

genic TCR that have repeatedly responded to the same

TCR-specific antigen were termed “memory cells”. This

approach appeared to limit the ongoing research on

the epigenetic component of immunological memory.

Doubts have been expressed about the extent to which

such models can accurately characterize real memory

cells that are formed during immunological memory

development. The spectrum of activation markers

and functionality of the transgenic T  cells reacting to

the same antigen could be only indirectly associated

with immunological memory [5].

The main obstacle was lack of the methods to

activate memory cells selectively that would facilitate

their cloning and molecular identification of TCR. One

important breakthrough was discovery of the ability

of memory cells to proliferate in response to allogene-

ic antigen-presenting cells (APC) exposed to an acute

heat shock (45°C, 1h) [6]. This feature made it possible

to induce a selective memory cell immune response,

which does not involve naive T  lymphocytes [7,  8].

This, in turn, provided the possibility for further cellu-

lar cloning of memory T lymphocytes, obtaining T

  cell

hybridomas from them, and identifying their T  cell

receptors. One of these receptors, named 1d1, which

is specific to the allogeneic histocompatibility mole-

cule class I (H-2Kb) presented on the EL-4 lymphoma

cells, was subjected to gene cloning and further ad-

vanced study. Retroviral transduction of the genes of

TCR chains into the thymoma 4G4 cells that do not

carry their own TCR and associated CD3 complexes

has shown that such transduction leads to expression

of the transgenic TCR 1d1 and appearance of the CD3

complex on the cell surface [9].

TRANSFER OF THE TCR β-CHAINS GENES –

CONSTRUCTION OF NEW MODELS

FOR STUDYING HOMEOSTATIC

PROLIFERATION OF T CELLS

The next step was to obtain animal lines with

expression of individual transgenic α- and β-chains

of TCR 1d1. Expression of β-chains occurs strictly ac-

cording to the rules of allelic exclusion. Therefore,

the β-chain transgene of the 1d1 receptor contributes

to suppression of gene rearrangement and prevents

expression of endogenous β-chains in the recipient

T  lymphocytes. The hCD2 promoter used in the work

of Silaeva et al. [10] initiates transgene expression at

the time of the start of gene rearrangements of en-

dogenous β-chains. By this time, there is a small per-

centage of T  cells, in which rearrangement of their

β-chain genes has already occurred and the transgene

expression is suppressed. As a result, the repertoire

of T  cells of the transgenic organism consists of two

parts: 10-20% of T  cells expressing the genes of their

own rearranged β-chains, and 80-90% of T

cells pro-

ducing the transgenic β-chain. A more detailed analy-

sis of the T  lymphocyte characteristics in mice with

the transgenic β-chain showed that the cells express-

ing the transgene are mainly represented by the na-

ive T  lymphocytes with the CD44

−

CD62L

phenotype,

whereas the T  lymphocytes expressing endogenous

TCR β-chains have the phenotype of activated effector

cells, CD44

CD62L

−

[10]. This could be easily explained

by the fact that survival and homeostasis of T cells

largely depend on receiving tonic signals via interac-

tion of their T cell receptors with the endogenous mol-

ecules of major histocompatibility complex (MHC) [11].

In other words, T  cells need a certain level of phys-

iological autoreactivity for survival and homeostasis

maintenance that prevents their death, just as in the

thymus, where interaction of thymocytes with their

own MHC/peptide complexes promotes their positive

selection and prevents their “death by neglect”. Lower

and upper limits of this physiological autoreactivity

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BIOCHEMISTRY (Moscow) Vol. 90 No. 2 2025

Fig.  1. A scheme of acquisition of the phenotype of naive or activated cells by a T lymphocyte, based on investigation of

animals with transgenic TCR β-chain. MHC, major histocompatibility complex; TCRs, T cell receptors.

range, likely, facilitate formation of the correspond-

ing phenotypes of naive and activated T  lymphocytes.

Decrease in the diversity of specificities in the TCRs

with transgenic β-chains should lead to the deficit in

receiving such tonic signals and increased competi-

tion of the T  cells for the available endogenous MHC

ligands. On the contrary, the T  lymphocytes that ex-

press a variety of normally rearranged endogenous

β-chains would be under conditions of relative abun-

dance, which could be compared to the conditions of

lymphopenia or immune response to an introduced

pathogenic microorganism multiplying within a body.

Our experiments revealed that the balance between

the naive and activated T  cells is maintained by their

interactions with endogenous MHC/peptide complexes;

it also means that the balance is mediated by auto-

reactivity of T  cells. Moreover, we can significantly

change this balance by influencing interactions of TCR

with the endogenous MHC/peptide complexes – in fa-

vor of naive cells if we limit the interaction, and in

favor of activated cells if we facilitate the interaction.

Thus, acquisition of the naive or activated phenotype

by a T  lymphocyte can be considered as a result of

availability of the corresponding MHC ligands. At the

same time, it became clear that acquisition of the

“activation phenotype” by the T  lymphocyte, which

is characteristic of memory cells, reveals its homeo-

static interaction with the MHC molecules of the cel-

lular environment, rather than actual “experience”

of interaction with a specific antigen. A hypothetical

scheme illustrating acquisition of naive and activated

cell phenotypes by a T  lymphocyte is shown in Fig. 1.

In this case, acquisition of the phenotype of activated

T  cells (CD44

CD62L

−

effectors or CD44

CD62L

mem-

ory cells) could be caused by interaction with the

specific antigen during an immune response, under

condition of lymphopenia caused by radiation, or by

significant reduction in the diversity of T  cell rep-

ertoire containing different TCRs built from the en-

dogenously rearranged α- and β-chains genes. Onthe

contrary, acquisition of the naive CD44

−

CD62L

phe-

notype could be associated with the lack of antigenic

stimulation or limited diversity of their TCR specificity

due to the invariant transgenic β-chain in their struc-

ture. It is also foreseeable that the proposed scheme

may be relevant for migration of naive T cells through

the lymph and bloodstream, and activated T  lym-

phocytes – through tissues and extravascular areas

of the body.

Β-chains are combined with normally rearranged

endogenous α-chains in both parts of the repertoire.

Since the “transgenic” part of the repertoire contains

an invariant β-chain, diversity of the T  cell receptors

in the transgenic animals is significantly reduced,

which leads to moderate suppression of immune re-

sponses to alloantigens and loss of the ability to reject

an allogeneic tumor. This was shown with the B10.

D2(R101) (KdIdDb) mouse line, which was used as

the genetic basis for expression of the 1d1 receptor

β-chain transgene [10]. Normally, T  lymphocytes of the

wild type mice of this line respond well in the mixed

lymphocyte reaction (MLR) to the stimulator cells iso-

lated from the C57BL/10 (H-2b) or FVB (H-2q) mice.

Transplantation of the EL-4 lymphoma cells originat-

ing from the C57BL/6 (H-2b) mice leads to the devel-

opment of a full-fledged immune response to the lym-

phoma cells and their complete rejection within 12-14

days, despite the fact that antigenic differences in the

transplant antigens in this case are represented only

by the allogeneic molecule H-2Kb. On the other hand,

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BIOCHEMISTRY (Moscow) Vol. 90 No. 2 2025

the response of T  lymphocytes from the transgenic

mice to the C57BL/10 (H-2b) and FVB (H-2q) stimula-

tor cells was reduced. When the EL-4 lymphoma cells

were injected into transgenic mice, the chronic im-

mune response developed, and the immune selection

of tumor cells occurred, followed by the loss of the

H-2Kb molecule, tumor growth, and, as a result, death

of animals 1-2 months after transplantation [12].

TRANSGENIC TCR β-CHAINS –

EFFECTS ON INTERACTION

OF T LYMPHOCYTES WITH TUMOR CELLS

Our research revealed that, despite the antigenic

differences between the tumor (H-2Kb) and the recip-

ient (H-2Kd) in one of the major histocompatibility

antigens, the tumor development process in the an-

imals with the transgenic TCR β-chain acquires char-

acteristics of chronic rejection. It occurs through the

stages of elimination, equilibrium, and escape of the

immune response. This escape is accompanied by the

loss of the specific antigen of tumor cells (the H-2Kb

histocompatibility molecule) in the course of the

process of resisting to the recipient immune system

and selection of the weakly immunogenic variants of

tumor cells under its pressure [12]. In other words,

the phenomenon of rejection of an allogeneic tumor

became a phenomenon of its progression under the

pressure of the immune system in the allogeneic re-

cipient due to the limited diversity of the repertoire

of T  cell receptors. Our data show that effectiveness

of the antitumor immune response could depend on

the range and diversity of the repertoire of T  cell re-

ceptors. Theoretically, this diversity is very high and

amounts to ~10

. Only a small part of it is realized,

since the total number of T  cells in the human body

is ~4-5  ×  10

[13, 14]. This means that the process of

overcoming of immunological defense by the tumor

and limited successes and achievements in the field of

tumor immunotherapy could be due to the deficiency

in the specificity of T cell receptors necessary for the

successful immunological recognition of tumor neoan-

tigens. It cannot be ruled out that the development

of approaches to artificially expand the repertoire

of receptors of cells of the adaptive immune system

could help to overcome this obstacle. This, in turn,

indicates that success in the development of tumor im-

munotherapy methods should be expected in the area

of delivering of the genes of T  cell receptors with re-

quired specificity to the patients, i.e., a type of passive

immunotherapy. Fundamental aspects of the revealed

patterns are equally important, as they shed light on

the nature of alloreactivity, a phenomenon that ap-

peared to be critically dependent on the diversity of

the T lymphocyte receptor repertoire in the recipient.

The obtained results support the hypothesis that the

T  lymphocyte receptors have an intrinsic ability to re-

act with any of the allelic variants of MHC molecules

[15]. According to it, the allogeneic immune response

is a combination of the responses of individual recip-

ient T  lymphocyte clones to foreign MHC/peptide com-

plexes that did not participate in the negative selec-

tion of T  lymphocytes in the thymus and formation of

their repertoire. On the other hand, negative selection

of T  cells in the thymus as a result of “strong” interac-

tion with their own MHC/peptide complexes could be

considered as the first and last allogeneic reaction of

the emerging repertoire of T  lymphocytes to the self

MHC molecules [16].

TRANSFER OF THE GENES

OF TCR α-CHAINS AND CHAIN-CENTRIC

T CELL RECEPTORS – PROSPECTS

FOR DEVELOPING NEW TECHNOLOGIES

OF IMMUNOLOGICAL PROTECTION

Transgenic T  cell receptors have become an in-

valuable tool for fundamental research on the mech-

anisms of immune system development and adaptive

immunity functioning. Availability of such research

tools for immunologists has provided opportunities

for visualizing the processes of intra-thymus selec-

tion of T  lymphocytes, formation of the ability of im-

mune system to distinguish between the “self” and

“non-self”, and of the processes of development and

differentiation of T  lymphocytes, and the mechanisms

of interaction of T  cell receptors with MHC/peptide

complexes [17].

A number of significant features of TCR biology

prevented the use of this tool in practice. These re-

ceptors are known to be heterodimers consisting of

α- and β- or δ- and γ-chains. The fate of the develop-

ing T  lymphocyte is strictly determined by the partic-

ular V, D, and J segments forming a functional gene,

the sequence of which will be in the reading frame.

During the intra-thymus development, rearrangement

of the gene segments of the β-, δ-, and γ-chains be-

gins first. Formation of the functional variant of the

β-chain gene blocks irreversibly further rearrange-

ments of other genes: the functional gene remains

active, while expression of recombinases and rear-

rangements of other β-, δ-, and γ-chains stops, and

they enter the region of condensed chromatin. At this

stage, a T  lymphocyte could become either an αβ- or

γδ-T cell; any such selection has a strictly alterna-

tive character [18]. Attempts to interfere with this

process and introduce a transgenic β-chain into the

T  lymphocyte negatively affect the immune system –

rearrangements of the genes of endogenous β-chains

stop. The mechanism of allelic exclusion is activated,

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BIOCHEMISTRY (Moscow) Vol. 90 No. 2 2025

which is accompanied by the dramatically reduced

diversity of T  cell receptors. As a result, most of the

receptors contain an externally introduced invari-

ant transgenic β-chain, which leads to suppression

of adaptive immunity [12, 19, 20]. Appearance of the

functional β-chain gene leads to expression of a gene,

protein product of which combines with the surrogate

α-chain, forming a pre-TCR localized in the endoplas-

mic reticulum that possesses an autocatalytic ability

to generate the signal for further T  lymphocyte de-

velopment. This signal initiates the second wave of

recombinase expression and rearrangement of the

α-chain genes, which can occur repeatedly by connect-

ing distally located V and J segments. The secondary

re-arrangement occurs if the previous combination

did not form a functional gene or the formed α-chain

is non-functional at the protein level, i.e., unable to

ensure the TCR interaction with MHC molecules of the

thymus medullary epithelium; as a result, the thymo-

cyte cannot pass the selection. Unlike β-chains, allelic

exclusion of α-chain genes is not strict, and their rear-

rangement could lead to formation of functional genes

on both chromosomes and, accordingly, to a T  lym-

phocyte with two αβ-T cell receptors [21]. Atthis time

point, the fate of the T lymphocyte depends critically

on whether the αβ heterodimer can interact with the

MHC class  II molecules located on the surface of the

thymus epithelium. Stable interaction of TCR with the

MHC class  II molecules mediated selection of the CD4

coreceptor by the developing T  cell. Weak and unsta-

ble interaction of TCR with the molecules of this type

triggers suppression of CD4 expression and commits

to the development towards T  cells with the CD8 core-

ceptor. Complete absence of interaction with the MHC

molecules results in the T  lymphocyte death, which is

called metaphorically “death by neglect”.

Nearly a decade ago, it became clear that the sig-

nificant part of the repertoire of T  cell receptors has

the property of chain-centricity, i.e., one of the TCR

chains is dominant in the recognition of MHC/peptide

complexes [22, 23]. Incorporation of the gene of the

single dominant-active chain of the chain-centric TCR

into the cell creates a unique possibility to influence

its interaction with the MHC/peptide complexes, thus

giving the T  cells a new specificity. It is worth noting

that modification of T  cells with the TCR α-chains has

many significant advantages. Due to the absence of al-

lelic exclusion of the α-chain genes and the possibility

of expressing two TCRα in one lymphocyte, transfer

of the α-chain gene could dramatically expand the

diversity of the repertoire of T  cell receptors without

causing systemic suppression of an immune response.

This possibility was realized by constructing the trans-

genic mouse line, in which α-chain of the chain-centric

TCR (1D1a) was incorporated in the wild-type mouse

genome as a part of the pTα cassette vector, which

provides tissue- and stage-specific expression of the

transgene. The transgenic α-chain originated from the

TCR T  cell hybridoma of memory cells specific to the

H-2Kb alloantigen of the EL-4 lymphoma cells. Com-

pared with the wild-type mice, the transgenic animals

had increased CD3 expression on the surface of dou-

ble negative thymocytes of the DN2 and DN3 stages,

undergoing the processes of gene rearrangement and

selection of functional β-chains. Moreover, increase

in the proportion of CD8

cells with the phenotype

of central memory cells (CD44

CD62L

) and effectors

(CD44

CD62L

−

) was observed in the peripheral lym-

phoid organs. Similarly to the effect of the transgenic

β-chain expression on the surface phenotype of T cells

described above, the data obtained in the analysis of

the 1D1a mice indicate an instructive role of the TCR

structure in the formation of the T  lymphocyte phe-

notypic properties.

The most unexpected finding was that, although

the transgenic mice expressed the α-chain of the origi-

nal TCR without its β-chain, they had developed immu-

nity to the EL-4 lymphoma cells. The animals rejected

the transplanted tumor cells within 2-3 days, which is

typical for the immunized animals with memory cells

for the immunizing antigen (the H-2Kb histocompati-

bility molecule). In MLR, the peripheral lymphocytes

of transgenic animals produced enhanced prolifera-

tive responses to the stimulator cells carrying H-2Kb

histocompatibility molecules on their surface. Thus,

the TCR structure, altered by the transgenesis of only

one α-chain, determined the functional properties of

T  cells in the transgenic organism. At the same time,

immune responses to third-party antigens in the 1D1a

mice were comparable to the wild-type animals. Thus,

expression of the transgenic TCR α-chain does not sup-

press immune responses of the 1D1a mice to other

antigens. During the study, we demonstrated that the

transgenic α-chain originating from the chain-centric

TCR can pair with endogenous β-chains and enter the

surface of T  lymphocytes in the form of heterodimers.

As a result, the repertoire of T  lymphocytes in the

transgenic animals is represented fully or partially by

the lymphocytes carrying an additional T  cell receptor

on the surface, the specificity of which is determined

by the transgenic α-chain [23].

These studies of the chain-centric TCR with domi-

nant active α-chains [23-26] provide a theoretical basis

for creating organisms with innate specific immuno-

logical resistance to certain pathogens.

CHAIN-CENTRIC T CELL RECEPTORS

AND ADOPTIVE IMMUNOTHERAPY

Studies of the 1D1a transgenic mice have shown

that these animals have enhanced innate antitumor

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BIOCHEMISTRY (Moscow) Vol. 90 No. 2 2025

Fig.  2. Gene transfer of the dominant active α-chain of the chain-centric TCR into recipient’s T lymphocytes or into the

zygote results in the repertoire of T lymphocytes with the additional TCR specificity. TCRs, T cell receptors.

immunity due to the expression of the dominant-ac-

tive α-chain of the chain-centric tumor-specific TCR.

Considering that transfer of the dominant α-chain

1D1a gene to the cells redirected the repertoire of

T  cells to fight tumor cells, a similar effect could be

expected with in  vitro modification of normal T  lym-

phocytes with the same dominant α-chain. Indeed, us-

ing retroviral transduction, we generated T  cells that

expressed 1D1a and showed protective and therapeu-

tic effects against the EL-4 lymphoma cells after adop-

tive transfer to the B10.D2(R101) mice [23]. Figure  2

shows how transduction or transfer of the α-chain

gene of the chain-centric TCR creates an additional

receptor and thereby modifies the specificity of the

recipient T  lymphocytes.

These results were confirmed with the infectious

model. The mouse memory T  cells for Salmonella ty-

phimurium antigens were generated in the invivo sys-

tem, with subsequent re-stimulation with antigens of

the same bacterium invitro. To identify the sequences

of TCR clonotypes that responded to re-stimulation, a

new-generation sequencing (NGS) method was used to

obtain repertoires of α-chains of the TCR in the mem-

ory T  cells stimulated by the model pathogen and in

the memory T  cells without stimulation. The α-chain

variants, frequency of which increased in the TCR

repertoire after re-stimulation with the Salmonella

antigens, were individually transduced into the nor-

mal T  lymphocytes. The dominant- active TCR α-chains

were identified by the ability of modified T  cells to

respond by proliferation to the Salmonella antigens

in  vitro. About 20% of the TCR repertoire of memory

cells that responded to Salmonella antigens exhibited

chain-centricity and contained the dominant-active

α-chain [24, 25]. Similar results were obtained in the

research investigating response to the Listeria mono-

cytogenes antigens [27].

Thus, chain-centric TCRs were found to comprise

a significant proportion of the T cell receptors that

respond to bacterial pathogen antigens. Moreover,

we developed an algorithm for the chain-centric re-

ceptor searching, according to which cloning of the

antigen-specific T  cells is not required. Since such

cloning is usually laborious and time-consuming, the

above-mentioned approach is quite desirable to be

applied in practice.

The dominant-active α-chains of the chain-cen-

tric T  cell receptors are promising candidates for the

use in adoptive immunotherapy of oncological and

infectious diseases, similar to the CAR-T chimeric an-

tigenic receptors used in oncology. Advantages of the

chain-centric TCR include the lack of immunogenic-

ity characteristic of the chimeric proteins and the

absence of the negative trans-completion effects on

the signal transmission into the T  lymphocyte, which

are unavoidable in the case of transduction of both

TCR chains. Moreover, it involves a simple and clear

search algorithm along with the possibility of direct

targeting of the immune response to the unique tumor

neoantigens [28, 29]. To assess feasibility of introduc-

tion of this approach into clinical practice, a preclin-

ical study was conducted assessing biosafety of the

T  cell products modified with the chain-centric TCRs

according to the following parameters: physiological

condition of animals, acute toxicity, immunotoxicity,

allergenicity, mutagenicity, tumorigenicity, and phar-

macokinetics. The conducted studies indicate the safe-

ty of the transgenic constructs injected with T  cells.

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BIOCHEMISTRY (Moscow) Vol. 90 No. 2 2025

At the same time, introduction of T  lymphocytes ac-

tivated during transduction in order to increase ef-

fectiveness of the procedure could lead to nonspe-

cific allergic reactions in the recipient  [30]. Studies

evaluating the safety of the transfer of chain-centric

receptors, characteristics of transgenic animals, and

their differences from the wild-type mice were also

conducted, and the functional condition of the im-

mune system of the 1D1a transgenic mice expressing

the dominant-active α-chain of the chain-centric TCR

specific to the H2-Kb molecule was analyzed. Neither

autoimmune diseases due to the random pairing of

transgenic TCRα with endogenous TCRβ variants nor

considerable disturbance of systemic homeostasis

were observed in the age dynamics of these mice. It

is worth noting that the TCRα transgene expression

could delay thymus involution and maintain the TCRβ

repertoire diversity in the old transgenic mice. Despite

the notable enhancement of the specific immune re-

sponse in the 1D1a mice, responses to the third-party

alloantigens were not affected, indicating that expres-

sion of the transgenic TCRα did not limit the immuno-

reactivity of the transgenic mice [26].

CHAIN-CENTRIC T CELL RECEPTORS

AND IMMUNOLOGICAL MEMORY

Along with the data accumulation in the research

field, there is a growing recognition of the fact that

chain-centricity is one of the basic properties of the

significant part of T  lymphocyte receptors. The ques-

tion arises: why this feature has not been investigat-

ed explicitly in the scientific literature over the last

half-century? Our analysis of the available sources

has shown that there are numerous indications of this

characteristic in the literature, but indirect in most of

the cases, fragmented, and not properly interpreted

[29]. The reason could be in the artificiality of the dis-

covered phenomenon since the transfer of the genes

of TCR chains does not occur in nature, and functional

value of the chain-centric receptors in the naturally

formed repertoire of T  cells is not clear. Nevertheless,

simultaneous expression of two TCR α-chains due

to successful rearrangement of the genes encoding

them on both chromosomes and incomplete allele ex-

clusion has been known, although, according to the

previous estimates, it is a rare event (1-10%) [31]. Re-

cent estimates of the frequencies of T  cell expressing

two T  cell receptors simultaneously are dramatically

higher and is ~16% [32,  33]. Proportion of these cells

increases considerably with age [34], as well as in

some autoimmune diseases [35]. In the acute phase

of the response to infection with lymphocytic chorio-

meningitis virus (LCMV), up to 60% of the virus-spe-

cific T  cells express double TCR. Moreover, after the

infection is complete, memory cells contain increased

frequencies of the CD4

T  cells with two TCRs [36].

It is also interesting that in the patients with Kawa-

saki syndrome who have undergone immunoglobulin

therapy, the proportion of the CD8

memory cells with

two TCRs increases [37].

Thus, simultaneous expression of two T  cell recep-

tors has a functional significance and is involved in

formation of immunological memory, but its relation-

ship with the effect of chain-centricity of TCR α-chains

is not obvious. Considering that we detected the first

chain-centric TCR in the long-lived memory cells, it

is likely that the frequencies of such TCRs are rath-

er low at the earlier stages of the immune response.

Presumably, this could be a reason why chain-centric

TCRs were not detected in the 1990s, at the peak of

investigations focused on the structure and functions

of TCR. At the moment, it is not entirely clear wheth-

er chain-centricity is an intrinsic feature of some

T  cell receptors or whether it is related to selection

of the repertoire of memory T  cells. Our work aimed

at identifying frequencies of the chain-centric TCR

during formation of immunological memory would

shed light on this fundamental issue. The results of

the T  cell repertoire study in the animals immunized

with allogeneic tumor cells confirm indirectly the hy-

pothesis of the chain-centric TCR expression mainly

by the memory cells. Using an experimental model

of induction of the antitumor immune response, se-

quential changes in characteristics of the TCR reper-

toire of T  cells involved in the primary and secondary

immune responses to allogeneic tumor antigens were

studied. Bioinformatics analysis of the TCR repertoire

showed that, compared with the primarily activated

effectors, re-stimulation of memory cells with the spe-

cific antigen led to their enrichment with the clono-

types that expressed TCR α-chains with high potential

cross-reactivity and the increased strength of interac-

tion with both MHC molecules and docked peptides.

No significant changes in the psycho-chemical char-

acteristics of TCRβ were observed in the reactivated

clonotypes of memory T cells, which may indicate

the dominant role of TCRα in the secondary alloge-

neic immune response. This effect could be due to

the increase in the frequencies of chain-centric TCRs

during the memory cells formation [38].

IMMUNOLOGICAL MEMORY

AND CYCLOPHILIN A

In the recent decades, extracellular cyclophilin A

and its biological activity have been objects of seri-

ous studies in our laboratory. Interest in this protein

first arose when T.  V.  Anfalova attempted to cultivate

in  vitro thymic T lymphocytes (thymocytes) of mice

KAZANSKY et al.168

BIOCHEMISTRY (Moscow) Vol. 90 No. 2 2025

treated with high doses of hydrocortisone, causing

apoptosis of more than 98% of thymocytes. A minor

part of the cells remained viable, proliferated in the

in  vitro culture, and secreted into the medium an

unknown factor with the properties of radioprotec-

tor, significantly prolonging life of the animals that

received sublethal doses of radiation. This effect was

found to be associated with the increased migration

of stem cells from the bone marrow of irradiated

animals to their peripheral lymphoid organs. It was

reproduced in  vitro: the factor of cortisone-resistant

thymocytes induced chemotaxis and migration of the

hematopoietic cells of different linages, including

T  cells, B  lymphocytes, monocytes, granulocytes, and

dendritic cells. On this basis, a test suitable for iso-

lating and purifying the target protein was developed.

Further purification and identification of the factor of

cortisone-resistant thymocytes via Edman degradation

allowed identifying cyclophilin  A [39]. Recombinant

production of the human cyclophilin  A with demon-

stration of its biological effects, partially reproducing

the effects of the factor of cortisone-resistant thymo-

cytes, confirmed our previous findings [40, 41]. Fur-

ther investigation of the cyclophilin A biological ac-

tivity attracted a lot of attention when its antitumor

and antimetastatic effects were revealed [42]. We have

shown pro-inflammatory effects of cyclophilin  A in

the study of expression of the genes of acute phase in-

flammatory proteins in the animal liver after admin-

istration of high doses of this protein [43]. Moreover,

cyclophilin  A, similar to the well-known proinflamma-

tory factor TNFα, appears to exhibit an embryotoxic

effect, disrupting embryonic development during or-

ganogenesis [44]. Thus, cyclophilin A is involved in

regulation of acute inflammation, but its role and

part in the regulation of this response has yet to be

established.

A wide range of studies have revealed the in-

volvement of the extracellular cyclophilin  A in in-

flammatory and autoimmune diseases. The available

data are presented and discussed in the reviews by

Kalinina et  al.  [45] and Xue et  al.  [46]. The function

of this protein in immunoregulation involves polar-

ization of the immune response towards the type  1

T  helper cells [47].

Two areas (pieces) of our studies, the investiga-

tion of memory cells and of cyclophilin A, which ap-

pear to be not closely related, suddenly fit the puz-

zle when we determined that the cortisone–resistant

thymocytes (producers of cyclophilin A [39]) contain

memory T  cells, comprising the major part of this cell

population. Administration of hydrocortisone at a dose

of 2.5  mg per animal to the pre-immunized animals

led to the 95-98% decrease in the thymus cellularity,

but at the same time to the multifold increase in the

proliferative response of the remaining lymphocytes

in the thymus to the immunizing antigen. This indi-

cated enrichment of the thymocyte population with

the mature cortisone-resistant lymphocytes with the

antigenic specificity [48]. Most likely, exposure to high

doses of hydrocortisone enriches the thymus with

T  lymphocytes that express high levels of antiapoptot-

ic molecules (which was repeatedly noted in memory

cells) [49, 50].

Cyclophilin  A is known to play an important

role in the negative regulation of apoptosis [51, 52].

Moreover, there are common features in the signal-

ing pathways involved in the memory cell differenti-

ation and regulation of oxidative stress by cyclophi-

lin  A [53, 54]. It was also shown in the recent study

that in the course of the signal transduction through

TCR, cyclophilin A regulates activity of the ZAP70 ty-

rosine kinase, which is important for activation and

differentiation of T  cells [55]. According to the data

available to date, the increased level of cyclophilin  A

appears to be part of the epigenetic profile of the

memory T  cells.

The reason and biological significance of the

memory T  cells emergence in the thymus remain

enigmatic. The thymus is traditionally considered to

be a central lymphoid organ focused on the export

of T  lymphocytes formed in it. The major function of

the thymus involves creation of a microenvironment

that ensures expression of RAG-1 and -2 recombinases

responsible for recombination of the TCR gene seg-

ments in T  lymphocytes. Therefore, memory T  cells

are assumed to migrate to thymus to edit α-chains

of their TCR, which would have the following conse-

quences: (i)  maintenance of the diversity of the T  lym-

phocyte receptor repertoire on the periphery due to

the changes in TCR of memory cells that formed in

excess during the immune response and became “un-

necessary” upon its completion; (ii)  formation of TCR

α-chains that ensure high affinity of the memory T  cell

receptors to the antigen. In other words, TCR editing

of a memory cell could lead to the formation of ei-

ther a receptor with a new specificity or a chain-cen-

tric TCR with an increased interaction affinity of its

α-chain to the original (immunizing) MHC/peptide

complexes [38]. Based on the data available to date,

we believe that the TCR editing, leading to the for-

mation of a chain-centric receptor, could be one of

the mechanisms of T  cell functional avidity matura-

tion, similar to the affinity maturation of the B-lym-

phocyte receptor. Figure  3 presents the hypothetical

scheme (based on the data results described in this

work) of the T  lymphocyte repertoire formation and

further transformations of T  lymphocytes during the

immune response. This scheme differs from the oth-

er models of linear differentiation of memory cells

from naive precursors into effectors and further into

memory cells, since it assumes formation of memory

IMMUNOLOGICAL MEMORY AND TRANSGENIC TCR 169

BIOCHEMISTRY (Moscow) Vol. 90 No. 2 2025

Fig. 3. Memory cells during the antigen-independent formation of the T cell repertoire in the immune response and its

completion. TCRs, T cell receptors.

cells regardless of the initial contact with the antigen.

This formation is determined by the TCR structure and

features of its interaction with the microenvironment

of the T  lymphocyte. In this case, there are T  cells,

which perform in the secondary immune response

functions that we call immunological memory. Our

experiments showed that T cells with the properties

of memory cells pre-exist before the contact with

the antigen. They appear to be “not visible” in the

primary immune response due to their small num-

ber and long time required for their antigen-specific

clones to grow to the experimentally detectable size.

They could also be detected in the primary response

if the α-chain transgene of the chain-centric TCR is

introduced into an organism. The presented scheme

of the memory cell formation, regardless of antigenic

stimulus, is in agreement with the existing ideas about

the constitutive formation of memory cells and early

determination of their fate [56, 57], which now has

direct experimental confirmation [23, 26].

Based on the results of our studies of the cor-

tisone-resistant thymocytes, we suggest that memory

cells could be producers of cyclophilin  A, which pro-

motes such “secondary selection” of T  lymphocytes in

the thymus.

Exhaustive evidence of this is not yet available,

but its exploration may be an important area of fur-

ther research. It has not been ruled out that cyclo-

philin  A could serve as a diagnostic marker for the

detection and identification of memory cells.

CONCLUSION

In general, it could be concluded based on the

results of our investigations that the relevant study

of immunological memory should be based on tech-

niques that provide an assessment of their specifici-

ty, functional properties, and protective effects. It has

been generally recognized that expression of the

genes of activation markers in T lymphocytes mostly

reflects general homeostatic interactions of the lym-

phoid system with the microenvironment rather than

an immune response to foreign antigens, and cannot

be directly attributed to the properties characterizing

cells of immunological memory. Memory cell cloning

with further identification of their TCRs allowed us

to reveal a relationship between the formation of im-

munological memory and accumulation of T  lympho-

cytes with chain-centric receptors. The notion about

such property of the part of TCRs has not been so

far fully clarified. Advances made during the study of

such receptors are promising for the development of

new methods of adoptive immunotherapy of cancer

and infectious diseases, as well as for creating organ-

isms with innate specific resistance to bacterial and

viral pathogens. The obtained data of the pilot study

indicate possible association between immunological

memory and cyclophilin  A production. In the future,

it seems very important to clarify the role of cyclo-

philin A in the formation and functioning of memo-

ry T  cells and to obtain data confirming or rejecting

KAZANSKY et al.170

BIOCHEMISTRY (Moscow) Vol. 90 No. 2 2025

the hypothesis implying the existence of the mecha-

nism of “secondary selection” in the memory cells in

the thymus.

Contributions. D.B.K., A.A.K., and L.M.Kh. partic-

ipated in the general discussion of the concept, ide-

ology, and work plan scheduling; D.B.K. prepared the

manuscript; A.A.K. and L.M.Kh. edited the manuscript

and performed its technical design.

Funding. This work was financially supported by

the Russian Science Foundation [grant no.22-15-00342

(2022-2024)].

Ethics approval and consent to participate. This

work does not contain any studies involving human

and animal subjects.

Conflict of interest. The authors of this work de-

clare that they have no conflicts of interest in finan-

cial or any other sphere.

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