Article

ISSN 0006-2979, Biochemistry (Moscow), 2024, Vol. 89, No. 5, pp. 853-861 © Pleiades Publishing, Ltd., 2024.

853

MINI-REVIEW

Reverse Genetics Applied to Immunobiology

of Tumor Necrosis Factor, a Multifunctional Cytokine

Sergey A. Nedospasov

1,2,a

*, Andrei A. Kruglov

, Alexei V. Tumanov

Marina S. Drutskaya

1,2

, Irina V. Astrakhantseva

, and Dmitry V. Kuprash

Division of Immunobiology and Biomedicine, Sirius University of Science and Technology,

354340 Federal Territory Sirius, Russia

Center for Precision Genome Editing and Genetic Technologies for Biomedicine,

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 119991 Moscow, Russia

Laboratory of Systems Rheumatology, German Rheumatism Research Center (DRFZ), a Leibniz Institute,

10117 Berlin, Germany

Department of Microbiology, Immunology and Molecular Genetics,

University of Texas Health Science Center at San Antonio, 79229 San Antonio, TX, USA

e-mail: sergei.nedospasov@gmail.com

Received November 21, 2023

Revised December 28, 2023

Accepted February 19, 2024

Abstract— Tumor necrosis factor (TNF) is one of many cytokines– protein molecules responsible for communi-

cation between the cells of immune system. TNF was discovered and given its grand name because of its striking

antitumor effects in experimental systems, but its main physiological functions in the context of whole organism

turned out to be completely unrelated to protection against tumors. This short review discusses “man-made”

mouse models generated by early genome-editing technologies, which enabled us to establish true functions

ofTNF in health and certain diseases as well as to unravel potential strategies for improving therapy of TNF-de-

pendent diseases.

DOI: 10.1134/S0006297924050067

Keywords: tumor necrosis factor, TNF, conditional mice models, gene knockout, reporter mice, gene overexpression

Abbreviations: ESCs, embryonic stem cells; LT,lymphotoxin; TNF,tumor necrosis factor; UTR,untranslated region.

* To whom correspondence should be addressed.

INTRODUCTION

Almost 50 years ago, Lloyd J. Old and his col-

leagues reported an interesting experiment performed

to study the antitumor effect of bacterial endotoxin

and other substances, which later would be known

as activators of innate immune receptors [1]. As it

was shown, combination of the infection with BCG

(Tb vaccine mycobacterial strain) and subsequent in-

jection of lipopolysaccharide (LPS) from Escherichia

coli leads to the appearance in blood serum of an un-

known “factor” with marked antitumor activity, which

was referred to as Tumor Necrosis Factor (TNF) [2].

This discovery was perceived as further development

of the ideas of William B. Coley, who pointed out the

possibility of using live bacteria or bacterial lysates in

the therapy for some tumors as early as at the begin-

ning of the 20th century [3]. Further studies showed

that TNF had a protein nature and could be produced

by the cells of immune system in response to vari-

ous stimuli, e.g., by myeloid cells in response to LPS.

Molecular cloning and heterologous expression of the

human and mouse Tnf genes made it possible to relate

antitumor activity to a single protein that could be de-

fined as a “cytokine”, i.e., the rapidly increasing super-

family of protein mediators of cell communications.

Administration of the recombinant TNF produced in

E.  coli to mice fully reproduced antitumor activity of

the native TNF[4], and its high doses resulted in man-

ifestation of toxicity associated with anacute inflam-

NEDOSPASOV et al.854

BIOCHEMISTRY (Moscow) Vol. 89 No. 5 2024

matory response in mice (toxicity was not observed in

the L. Old’s experiments [2], probably because it was

impossible to achieve the same high concentrations as

in the experiments with recombinantTNF).

TNF has a molecular weight of 17  kDa; however,

in order to perform its physiological (including anti-

tumor) functions, it has to form a homotrimer, which

is a high-affinity ligand of two different receptors:

TNFR1(p55) and TNFR2(p75)[5]. These receptors dif-

fer both in the type of transmitted intracellular signal

and in the patterns of tissue-specific expression.

Following the molecular description of TNF and

its receptors in the 1980s, other members of the large

families of TNF-like cytokines (most of which are

membrane-bound molecules) and TNFR-like receptors

were soon discovered; they are able to transmit sev-

eral types of intracellular signals, thereby determin-

ing biological activity of the ligands of this family[6].

There were hundreds of papers describing TNF activi-

ty in different situations in  vitro and in vivo; however,

physiological function of TNF in the context of a whole

organism was established in 1990s mainly through

two strategies of reverse genetics: creating mice with

the Tnf gene knockout and generating transgenic mice

with its overexpression. Moreover, several interesting

mouse lines were isolated by the methods of direct ge-

netics. Below we will discuss numerous mouse models

that have been independently created and character-

ized in various laboratories, as well as the so-called

“reporter” mice, which represent additional tools for

studying immunobiology and physiology of this inter-

esting cytokine.

MICE WITH COMPLETE, PARTIAL

OR REGULATED GENETIC DEFICIENCY OF TNF

The knockout techniques that appeared before the

era of genomic nucleases and became widespread in

the 1980s were based on targeting the genes in mouse

embryonic stem cell lines (ESCs) (for other animals,

such lines were created much later). Due to the low ef-

ficiency of this procedure in the mouse ESCs it became

necessary to introduce antibiotic resistance markers to

select knockout clones (usually neomycin-resistance);

as a result, the expression cassette containing the re-

sistance gene (the so-called “neo-cassette”) remained

forever in the structure of the targeted locus and could

influence the activity of the nearby genes. Noteworthy,

it took 10 years from the cloning of the mouse Tnf gene

to the creation of the first Tnf knockout mice. At least

four laboratories obtained such mice almost simulta-

neously and independently (Table  1), but the palm of

victory obviously went to the laboratory of G. Kollias

in Greece. When creating the Tnf knockout mice, one

had to take into consideration two significant peculiar-

ities of this gene. Firstly, the Tnf gene is located very

close to the related lymphotoxin genes Ltα and Ltβ[7];

therefore, combined Tnf/Lt knockouts cannot be ob-

tained by crossbreeding of mice with single knockouts.

Secondly, the Tnf/Lt locus is within the major histo-

compatibility complex (MHC)[7]; hence, it is very diffi-

cult to obtain congenic mouse lines with modifications

of the Tnf gene and the required alleles of the MHC

genes, so that the knockout mice should be designed

with particular reference to the genetic basis of ESCs

and mouse lines used for subsequent backcrossing.

Pasparakis et al. [8] described new TNF functions

associated with its role in the structural and func-

tional organization of lymphoid tissues, about which

the discoverers of TNF had no idea. It should be not-

ed that the related cytokine, LTα, was considered for

10years to be a functional analog of TNF, produced by

lymphoid rather than by myeloid cells. This view on

lymphotoxin was supported by the fact that the re-

combinant protein demonstrated an antitumor activity

similar to that of TNF in the model of transplantable

sarcoma in mice, as well as in cell cultures sensitive

to cytotoxic effects of TNF [20]. These results indicat-

ed potential redundancy in TNF and LTα functions, in-

spiring creation of a double knockout of both genes.

Table  1 presents some mouse lines with such double

knockouts. Note that earlier the lymphotoxin gene

knockout produced a sensational phenotype [21]: in

mice peripheral lymphoid organs were completely

absent, with the exception of spleen. Further compar-

ison of the phenotypes in the knockout mice made it

possible to differentiate between the functions of TNF

and lymphotoxin, e.g., in the case of organogenesis of

Peyer’s patches[22], which may be explained by signal

transduction from the membrane lymphotoxin com-

plex LTα

/LTβ

 [23] through LTβR [24], while a minor

overlap in functions is due to the fact that the soluble

lymphotoxin(LTα

) is able to trigger the signaling cas-

cade in  vivo through TNFR1 and TNFR2[17].

The technique of conditional (tissue-specific or in-

ducible) genetic recombination developed in the early

1990s in mammalian cell culture[25] and then in the

cells of transgenic[26,27] and knockout [28-30] mice

was based on LoxP/Cre recombination system of bacte-

riophage  P1, has truly revolutionized biological stud-

ies. It became possible, firstly, to circumvent the prob-

lem of embryonic lethality typical for a great number

of genes, even those with unknown non-redundant

functions in embryogenesis, and, secondly, to relate

particular functions of the genes and their products to

particular types of producer cells. This strategy proved

to be especially fruitful for studying the genes whose

products manifested pleiotropic functions, which is

typical for cytokines and other regulatory molecules.

Development of a panel of conditional mouse

lines for the Tnf gene began almost 20 years ago[14],

REVERSE GENETICS APPLIED TO TNF IMMUNOBIOLOGY 855

BIOCHEMISTRY (Moscow) Vol. 89 No. 5 2024

Table 1. Mouse models with TNF deficiency

Genotype of the Tnf/Lt locus Description References

Mice with the complete TNF gene knockout generated by reverse genetics

Tnf

–/–

complete knockout created by targeting in ESCs of the EK.CCE line

of mice of the 129/Sv lineage, with one backcrossing at C57BL/6

[8]

Tnf

–/–

complete knockout created by targeting in ESCs of the W9.5 line

from mice of the 129/Sv line, with one backcrossing at C57BL/6

[9]

Tnf

–/–

complete knockout created by targeting in ESCs from the hybrid

of the first generation of C57BL/6 and CBA mice, with selection

of the Tnf gene knockout in the C57BL/6 haplotype and 5 rounds

of backcrossing at C57BL/6

[10,11]

Tnf

–/–

complete knockout created by targeting in ESCs from the BL/6-III line

on the genetic basis of C57BL/6

[12]

Tnf

–/–

derived from the mouse line created for conditional targeting of TNF

on the genetic basis of 129/Sv, with multiple backcrossing atC57BL/6;

the targeted locus does not contain neo-cassettes

[13,14]

Mouse models with combined TNF/LT knockout

Tnf

–/–

Lta

–/–

(TNF/LTα double KO)

complete knockout of the Lta and Tnf genes created by targeting

in ESCs of the BL/6-III line on the genetic basis of C57BL/6

[12]

Tnf

–/–

Lta

–/–

(TNF/LTα double KO)

complete knockout of the Lta and Tnf genes created by targeting

in ESCs from the mouse129/Sv line, with one backcrossing atC57BL/6

[15]

Tnf

–/–

Lta

–/–

(TNF/LTα doubleKO)

complete knockout of the Lta and Tnf genes created by targeting

in ESCs from the mouse 129/Sv line, with one backcrossing at C57BL/6

[16]

Tnf

–/–

Ltb

–/–

(TNF/LTβdoubleKO)

complete knockout of the Ltb and Tnf genes created by targeting in ESCs

from the mouse 129/Sv line, with multiple backcrossing at C57BL/6

[17]

Tnf

–/–

Lta

–/–

Ltb

–/–

(TNF/LTα/LTβtriple KO)

complete knockout of the Lta, Tn, and Ltb genes created by targeting in

ESCs from the mouse129/Sv line, with multiple backcrossing at C57BL/6

[18]

Hypomorphic allele of the Tnf gene selected with the help of direct genetics

Tnf

PanR1/PanR1

, Tnf

PanR1/+

in the random mutagenesis experiment using N-ethyl-N-nitrosourea,

a dominant-negative mutant with the P138T amino acid substitution

in the mature TNF protein was found; the mutation prevents TNF

binding to the receptor TNFR1

[19]

Basic “platform” for generating conditional deletions of the Tnf gene in mice

Tnf

flox/flox

using genetic knockout in ESCs from the mouse 129/Sv line,

with multiple backcrossing at C57BL/6, the Tnf gene is framed by LoxP

sites (“floxed”); the modification does not affect activity of the gene

but allows for its subsequent elimination by Cre recombinase

in particular types of cells and/or in inducible fashion

[14]

first for the major populations of immunocytes: my-

eloid cells and lymphocytes. Using these mouse mod-

els, it was shown that the particular homeostatic or

pathogenic functions of TNF are associated with the

particular type of cells producing this cytokine. For ex-

ample, myeloid cells (primarily macrophages and neu-

trophils) are necessary for protection against intra-

cellular infections and formation of granulomas but,

NEDOSPASOV et al.856

BIOCHEMISTRY (Moscow) Vol. 89 No. 5 2024

at the same time, proved to be the major sources of

systemic TNF in different pathological states, e.g.,

LPS-induced toxicity [14]. On the other hand, TNF

produced by T and B cells performs homeostatic func-

tions, including organization of lymphoid tissue [31].

Later, mice with the constitutive or induced deletion

of the Tnf gene in dendritic cells, monocytes, lympho-

cytes[32-34], basophiles, microglia, mast cells[35], ep-

ithelial cells[36, 37], smooth muscle cells[38,  39], etc.,

were created. Interestingly, in all cases there were

unique phenotypic traits associated with the TNF pro-

duction by a particular cellular source.

MOUSE MODELS

BASED ON TNF OVEREXPRESSION

AND/OR “HUMANIZATION”

The first and the best-known transgenic mouse

system utilized the mechanism of posttranscriptional

regulation of the Tnf gene [40], confirmed the hypoth-

esis of a relationship between the overexpression of

TNF and the development of arthritis [41] and be-

came a widespread preclinical model for studying TNF

blockers. It was created in the Kollias’s laboratory[42]

in 1991. This model was an important addition to the

clinical results obtained by Mainiet al. [43] and Feld-

mann et al. [44] and made it possible to substantiate

anti-TNF therapy as an innovative strategy for treating

rheumatoid arthritis. In the original work of Kollias’s

team[42], the number of transgene insertions in mice

was several dozens, which resulted in manifestation

of the TNF-dependent polyarthritis in all mice at the

age of several months. Therefore, the drawbacks of

this model included the early development of only one

type of the TNF-dependent diseases, preventing mod-

eling of other autoimmune diseases. Later, the same

genetic construct was used to create transgenic mice

with a smaller number of transgenic insertions, mak-

ing it possible to ameliorate the pathogenic phenotype

and to extend the range of applications of this preclini-

cal model [45](Table2).

Note that in the works by Keffer et al. [42] and

Hayward et al. [45] the human Tnf gene was overex-

pressed in mice; at the same time, the mouse Tnf gene

was not deleted and could be expressed, while both

TNF receptors remained murine. In essence, these

were partially “humanized” preclinical models allow-

ing treatment of the experimental TNF-mediated pa-

thologies with involvement of the human TNF blockers,

most of them (with the exception of etanercept) being

species-specific and not working against mouse TNF.

Table 2. Mice with TNF overexpression

Mouse model Description References

Tgl278

transgenic line with the human Tnf gene under control of a strong promoter,

with modification of 3′-untranslated region(UTR), which resulted in high

expression of human TNF and caused severe polyarthritis in mice;

preclinical model for studying the effects of TNF blockers invivo

[42]

B6.Cg-Tg(TNF)#Xen

the same but with moderate levels of human TNF expression;

improved preclinical model

[45]

BPSM1

the line with spontaneous insertion of retrotransposon

in the mouse 3′-UTR Tnf, impairing the previously unknown mechanism

of posttranscriptional regulation;

TNF overexpression in this mouse line leads not only to severe

polyarthritis but also to heart valve disease

[46]

TNF

del4

, TNF

del5

, TNF

del6

and their combinations

the panel of mouse lines with impairment of three regions

in the 3′-UTR of the mouse Tnf gene regulating mRNA stability;

it has been shown that impairment of this system may result

in the extremely high TNF production causing death of some embryos

[47]

TNF

ΔARE

mice with deletion of the AT-rich region in 3′-UTR

of the mouse Tnf gene by the LoxP/Cre technique;

phenotypically similar to the Tgl278 and BPSM1 line but, in addition

to arthritis, developed inflammation in the ileum of the small intestine

[48]

Transgenic mice

with the entire human

Tnf/ltα/ltβ-locus

all three genes are under control of the intrinsic promoters/enhancers;

there was a moderate overexpression of genes of the human Tnf/Ltα/Ltβ locus;

cortical atrophy of the thymus

[49]

REVERSE GENETICS APPLIED TO TNF IMMUNOBIOLOGY 857

BIOCHEMISTRY (Moscow) Vol. 89 No. 5 2024

Table 3. Reporter mice for visualization of TNF expression

Genetic name of mouse model Description References

TNF-2A-Kat (B6.FRFPK+)

bicistronic expression of TNF and fluorescent protein Katushka

using the viral 2A peptide sequence

[58,59]

hTNF.LucBAC

recombination with the human TNF BAC clone, which resulted

in substitution of the luciferase gene for exon1 of the Tnf gene

[60]

FVB/N-Tg(CAG-EGFP,-Tnf)1Kul/J

mice with the “floxed” eGFP cassette,

which prevents transcription of the mouse Tnf cDNA;

TNF is expressed only after Cre-mediated recombination

[61]

The more advanced “humanized” model included mice

with the human Tnf gene inserted precisely at the site

of the mouse Tnf gene (genetic “knock-in”) and with

conservation of all regulatory elements [50]. These

mice did not demonstrate any abnormalities in the

development of lymphoid tissue microarchitecture; at

the same time, the human TNF performed protective

function in the case of intracellular infections, which

was indicative of normal in  vivo signaling through

the mouse TNFR1 receptor. On the other hand, the hu-

man TNF is unable to effectively activate the mouse

TNFR2[51], which was a disadvantage of this human-

ized model. For quite a number of experimental pa-

thologies (arthritis, acute hepatotoxicity, etc.) such lim-

itation of the model is not a problem, because most of

the biological effects of TNF are mediated by TNFR1.

Nevertheless, in some cases, TNFR2 may play a signif-

icant role; therefore, the mice with “humanization” of

both TNF and the extracellular domain of TNFR2 with

the possibility of conditional activation of TNFR2 were

developed to eliminate this limitation and to specify

the role of TNFR2 [52]. Transgenic mice expressing

Cre recombinase in FoxP3-positive cells were used to

demonstrate that signal transduction via TNFR2 in the

T-regulatory cells contributes to protection of the cen-

tral nervous system in the model of autoimmune pa-

thology[52]. One more interesting line contained the

modified Tnf gene, the product of which had certain

amino acid variations and, as a result, almost no sol-

uble TNF was formed[53]. The experiments with this

mouse line have demonstrated for the first time that

many TNF functions in vivo are mediated by the mem-

brane-bound but not soluble form.

Several mouse models with overexpression of the

endogenous mouse TNF due to impaired 3′-UTR re-

gions of the Tnf gene, which are responsible for mRNA

stability, were described(Table  2). In the BPSM1 mice,

there was spontaneous integration of retrotransposon

in the 3′-UTR[46] leading to the development of severe

polyarthritis, as well as heart valve disease. The panel

of mouse lines with combinatorial damage to several

regions of 3′-UTR involved in the control of mRNA sta-

bility demonstrates not only polyarthritis and heart

diseases but, in some cases, also embryonic lethality

as a result of extremely high TNF production  [47]. All

these regions are affected by the Cre/LoxP-mediated

deletion in the TNF

ΔARE

line developed in the Kollias’s

laboratory [48] 25years ago; therefore, with regard to

arthritis this model is equivalent to the BPSM1 line and

resembles the Tgl278 line, but with more pronounced

symptoms of systemic inflammation.

Taking into consideration complexities in regu-

lation of the genes encoding TNF and lymphotoxins,

detailed elucidation of the mechanism of accelerated

atrophy of the thymus in the mice carrying the entire

human locus Tnf/Lt as a transgene insertion (Table  2)

might need further investigation[49].

“REPORTER” MICE ARE AN IMPORTANT TOOL

FOR STUDYING TNF FUNCTIONS

Genetic constructs encoding different luminescent

or fluorescent proteins considerably expanded the pos-

sibilities of detection and visualization of gene activity

in  vitro and in vivo[54]. Transgenic mice, with expres-

sion of the protein under study being accompanied

by expression of the reporter protein, have become a

widespread tool for such studies [55]. In most cases,

transgenesis utilized bicistronic constructs that control

constitutive or conditional expression of the studied

protein and the fluorescent reporter protein. However,

it is also possible to design more complex constructs

that allow monitoring cells of a particular lineage of

cellular differentiation, with the fluorescence of one

protein switching to the florescence of another protein

in the course of differentiation[56].

Several variants of the reporter mice for TNF stud-

ies are known (Table  3). Luciferase, green fluorescent

protein GFP, and far-red fluorescent protein Katushka

were used as reporter proteins [57]. Detection of re-

porter proteins is possible not only by flow cytometry

and histological identification of TNF-producing cells,

but also by in vivo visualization.

NEDOSPASOV et al.858

BIOCHEMISTRY (Moscow) Vol. 89 No. 5 2024

CONCLUSIONS

TNF proved to be a surprisingly complex cytokine

with numerous functions both in homeostasis of the im-

mune system and in some nonimmune organs as well

as in different pathologies. Mouse models have made

it possible to link the protective and homeostatic func-

tions of TNF and its proinflammatory effects (which

may facilitate the development of certain pathologies),

to the particular types of the TNF-producing cells. One

conclusion that can be drawn from a large number

of studies discussed in this review is that the system-

ic TNF blockade in vivo will inevitably demonstrate

side effects due to neutralization of homeostatic and

protective functions of TNF. Consequently, elucidating

how to neutralize TNF only from a “pathogenic” cellu-

lar source[62], may allow us to apply improved thera-

peutic strategies to a variety of TNF-dependent diseases.

Acknowledgments. The authors are grateful to their

colleagues S.  I.  Grivennikov, L.  Tessarollo, A.  A.  Kuchmiy,

A.  R.  Galimov, S.  V.  Kozlov, I.  R.  Mufazalov, and R.  Nau-

mann for their contributions to creation and study of

some mouse models discussed in this review.

Contributions. S.A.N. the concept; S.A.N., I.V.A.,

and D.V.K. writing the manuscript; A.A.K., A.V.T., and

M.S.D. editing the manuscript and making important

additions. All authors participated in creation or study

of the mouse lines discussed in the review.

Funding. The work was supported by the Ministry

of Science and Higher Education of the Russian Feder-

ation, project “Development of Bioresource Collection

‘Collection of Laboratory Rodents with SPF-status for

Fundamental, Biomedical and Pharmacological Re-

search’ of the Institute of Biological Chemistry, Russian

Academy of Sciences” (agreement no.075-15-2021-1067).

The study of the phenotype of some genetically modi-

fied mouse lines was supported by the Russian Science

Foundation (project no.19-75-30032).

Ethics declarations. All manipulations were

carried out in accordance with the guideline of the

Federation of European Laboratory Animals Science

Associations. The experiments were approved by the

Bioethics Committee of the Institute of Molecular Biol-

ogy, Russian Academy of Sciences. The authors of this

work declare that they have no conflicts of interest.

REFERENCES

1. Parr, I., Wheeler, E., and Alexander, P. (1973) Similar-

ities of the anti-tumour actions of endotoxin, lipid A

and double-stranded RNA, Br. J. Cancer, 27, 370-389,

doi:10.1038/bjc.1973.45.

2. Carswell, E. A., Old, L. J., Kassel, R. L., Green, S.,

Fiore, N., and Williamson, B. (1975) An endotoxin-

induced serum factor that causes necrosis of tumors,

Proc. Natl. Acad. Sci. USA, 72, 3666-3670, doi:10.1073/

pnas.72.9.3666.

3. Coley, W. B. (1910) The treatment of inoperable

sarcoma by bacterial toxins (the mixed toxins of

the Streptococcus erysipelas and the Bacillus pro-

digiosus), Proc. R. Soc. Med., 3, 1-48, doi: 10.1177/

003591571000301601.

4. Pennica, D., Nedwin, G. E., Hayflick, J. S., Seeburg,

P.H., Derynck,R., Palladino, M.A., Kohr, W.J., Aggar-

wal, B. B., and Goeddel, D. V. (1984) Human tumour

necrosis factor: precursor structure, expression and

homology to lymphotoxin, Nature, 312, 724-729,

doi:10.1038/312724a0.

5. Brenner, D., Blaser, H., and Mak, T. W. (2015) Regu-

lation of tumour necrosis factor signalling: live or

let die, Nat. Rev. Immunol., 15, 362-374, doi: 10.1038/

nri3834.

6. Locksley, R. M., Killeen,N., and Lenardo, M. J. (2001)

The TNF and TNF receptor superfamilies, Cell, 104,

487-501, doi:10.1016/S0092-8674(01)00237-9.

7. Nedospasov, S. A., Shakhov, A. N., Turetskaya, R. L.,

Mett, V.A., and Georgiyev, G.P. (1985) Molecular clon-

ing of the genes encoding human tumor necrosis fac-

tors: tandem localization of the alpha and beta genes

in a short segment (6 k.b.p.) of the human genome,

Dokl. Akad. Nauk SSSR, 285, 1487-1490.

8. Pasparakis, M., Alexopoulou, L., Episkopou, V., and

Kollias,G. (1996) Immune and inflammatory respons-

es in TNF alpha-deficient mice: a critical requirement

for TNF alpha in the formation of primary Bcell fol-

licles, follicular dendritic cell networks and germinal

centers, and in the maturation of the humoral immune

response, J. Exp. Med., 184, 1397-1411, doi: 10.1084/

jem.184.4.1397.

9. Marino, M. W., Dunn, A., Grail,D., Inglese, M., Nogu-

chi,Y., and Richards,E. (1997) Characterization of tu-

mor necrosis factor-deficient mice, Proc. Natl. Acad.

Sci. USA, 94, 8093-8098, doi:10.1073/pnas.94.15.8093.

10. Taniguchi,T., Takata,M., Ikeda,A., Momotani,E., and

Sekikawa,K. (1997) Failure of germinal center forma-

tion and impairment of response to endotoxin in tu-

mor necrosis factor alpha-deficient mice, Lab. Invest.,

77, 647-658.

11. Iraqi, F., Sekikawa, K., Rowlands, J., and Teale, A.

(2001) Susceptibility of tumour necrosis factor-alpha

genetically deficient mice to Trypanosoma congolense

infection, Paras. Immunol., 23, 445-451, doi: 10.1046/

j.1365-3024.2001.00401.x.

12. Körner,H., Cook,M., and Riminton, D.S. (1997) Distinct

roles for lymphotoxin-α and tumor necrosis factor in

organogenesis and spatial organization of lymphoid

tissue, Eur. J. Immunol., 27, 2600-2609, doi: 10.1002/

eji.1830271020.

13. Kuprash, D. V., Tumanov, A. V., Liepinsh, D. J., Lem-

ckert, F. A., Hoek, R. M., Ledermann, B., Köntgen, F.,

REVERSE GENETICS APPLIED TO TNF IMMUNOBIOLOGY 859

BIOCHEMISTRY (Moscow) Vol. 89 No. 5 2024

De St Groth, B.F., and Sedgwick, J.D. (2005) Novel tu-

mor necrosis factor‐knockout mice that lack Peyer’s

patches, Eur. J. Immunol., 35, 1592-1600, doi: 10.1002/

eji.200526119.

14. Grivennikov, S.I., Tumanov, A.V., Liepinsh, D.J., Kru-

glov, A. A., Marakusha, B.I., Shakhov, A.N., Muraka-

mi,T., Drutskaya, L.N., Förster,I., Clausen, B.E., Tes-

sarollo,L., Ryffel,B., Kuprash, D.V., and Nedospasov,

S. A. (2005) Distinct and nonredundant in vivo func-

tions of TNF produced by T cells and macrophages/

neutrophils, Immunity, 22, 93-104, doi: 10.1016/

j.immuni.2004.11.016.

15. Eugster, H-P., Muller, M., Karrer, U., Car, B. D., Schny-

der, B., Eng, V. M., Woerly, G., Hir, M. L., Padova,

F.D., Aguet, M., Zinkernagel, R., Bluethmann,H., and

Ryffel, B. (1996) Multiple immune abnormalities in

tumor necrosis factor and lymphotoxin-α double-

deficient mice, Int. Immunol., 8, 23-36, doi: 10.1093/

intimm/8.1.23.

16. Amiot,F., Bellkaid,Y., Lebastard,M., Ave,P., Dautry,F.,

and Milon, G. (1996) Abnormal organisation of the

splenic marginal zone and the correlated leukocytosis

in lymphotoxin-alpha and tumor necrosis factor alpha

double deficient mice, Eur. Cytokine Netw., 7, 733-739.

17. Kuprash, D. V., Alimzhanov, M. B., Tumanov, A. V.,

Anderson, A. O., Pfeffer, K., and Nedospasov, S. A.

(1999) TNF and lymphotoxin beta cooperate in the

maintenance of secondary lymphoid tissue microar-

chitecture but not in the development of lymph nodes,

J. Immunol., 15, 6575-6580, doi: 10.4049/jimmunol.

163.12.6575.

18. Kuprash, D. V., Alimzhanov, M. B., Tumanov, A. V.,

Grivennikov, S. I., Shakhov, A. N., Drutskaya, L. N.,

Marino, M. W., Turetskaya, R. L., Anderson, A. O.,

Rajewsky,K., Pfeffer,K., and Nedospasov, S.A. (2002)

Redundancy in tumor necrosis factor (TNF) and lym-

photoxin (LT) signaling in vivo: mice with inactiva-

tion of the entire TNF/LT locus versus single-knockout

mice, Mol. Cell. Biol., 22, 8626-8634, doi:10.1128/MCB.

22.24.8626-8634.2002.

19. Rutschmann, S., Hoebe, K., Zalevsky, J., Du, X.,

Mann, N., Dahiyat, B. I., Steed, P., and Beutler, B.

(2006) PanR1, a dominant negative missense allele

of the gene encoding TNF-α (Tnf), does not impair

lymphoid development, J. Immunol., 176, 7525-7532,

doi:10.4049/jimmunol.176.12.7525.

20. Gray, P. W., Aggarwal, B. B., Benton, C. V., Bringman,

T.S., Henzel, W.J., Jarrett, J.A., Leung, D.W., Moffat,B.,

Ng,P., Svedersky, L.P., Palladino, M.A., and Nedwin,

G. E. (1984) Cloning and expression of cDNA for hu-

man lymphotoxin, a lymphokine with tumour necrosis

activity, Nature, 312, 721-724, doi:10.1038/312721a0.

21. De Togni, P., Goellner, J., Ruddle, N.H., Streeter, P. R.,

Fick, A., Mariathasan, S., Smith, S. C., Carlson, R.,

and Shornick, L. P. (1994) Abnormal development

of peripheral lymphoid organs in mice deficient in

lymphotoxin, Science, 264, 703-707, doi: 10.1126/

science.8171322.

22. Gogoleva, V. S., Kuprash, D.V., Grivennikov, S. I., Tu-

manov, A. V., Kruglov, A. A., and Nedospasov, S. A.

(2022) LTα, TNF, and ILC3 in Peyer’s patch organogen-

esis, Cells, 11, 1970, doi:10.3390/cells11121970.

23. Browning, J., Ngam-ek, A., Lawton, P., DeMarinis, J.,

Tizard, R., Chow, E. P., Hession, C., O’Brine-Greco, B.,

Foley, S.F., and Ware, C.F. (1993) Lymphotoxin beta, a

novel member of the TNF family that forms a hetero-

meric complex with lymphotoxin on the cell surface,

Cell, 72, 847-856, doi:10.1016/0092-8674(93)90574-A.

24. Crowe, P. D., VanArsdale, T. L., Walter, B. N., Ware,

C. F., Hession, C., Ehrenfels, B., Browning, J. L., Din,

W. S., Goodwin, R. G., and Smith, C. A. (1994) A lym-

photoxin-β-specific receptor, Science, 264, 707-710,

doi:10.1126/science.8171323.

25. Sauer,B., and Henderson, N. (1988) Site-specific DNA

recombination in mammalian cells by the Cre recom-

binase of bacteriophageP1, Proc. Natl. Acad. Sci. USA,

85, 5166-5170, doi:10.1073/pnas.85.14.5166.

26. Lakso,M., Sauer,B., Mosinger,B., Lee, E.J., Manning,

R.W., Yu, S.H., Mulder, K.L., and Westphal,H. (1992)

Targeted oncogene activation by site-specific recombi-

nation in transgenic mice, Proc. Natl. Acad. Sci. USA,

89, 6232-6236, doi:10.1073/pnas.89.14.6232.

27. Orban, P. C., Chui, D., and Marth, J. D. (1992) Tis-

sue- and site-specific DNA recombination in trans-

genic mice, Proc. Natl. Acad. Sci. USA, 89, 6861-6865,

doi:10.1073/pnas.89.15.6861.

28. Gu, H., Zou, Y. R., and Rajewsky, K. (1993) Indepen-

dent control of immunoglobulin switch recombina-

tion at individual switch regions evidenced through

Cre-loxP-mediated gene targeting, Cell, 73, 1155-1164,

doi:10.1016/0092-8674(93)90644-6.

29. Kühn, R., Schwenk, F., Aguet, M., and Rajewsky, K.

(1995) Inducible gene targeting in mice, Science,

269, 1427-1429, doi:10.1126/science.7660125.

30. Gu, H., Marth, J. D., Orban, P. C., Mossmann, H., and

Rajewsky, K. (1994) Deletion of a DNA polymerase

beta gene segment in T cells using cell type-specif-

ic gene targeting, Science, 265, 103-106, doi: 10.1126/

science.8016642.

31. Tumanov, A.V., Grivennikov, S.I., Kruglov, A.A., Sheb-

zukhov, Y.V., Koroleva, E.P., Piao,Y., Cui, C.-Y., Kuprash,

D.V., and Nedospasov, S.A. (2010) Cellular source and

molecular form of TNF specify its distinct functions

in organization of secondary lymphoid organs, Blood,

116, 3456-3464, doi:10.1182/blood-2009-10-249177.

32. Wolf, Y., Shemer, A., Polonsky, M., Gross, M., Mild-

ner, A., Yona, S., David, E., Kim, K.-W., Goldmann, T.,

Amit, I., Heikenwalder, M., Nedospasov, S., Prinz, M.,

Friedman, N., and Jung, S. (2017) Autonomous TNF

is critical for in vivo monocyte survival in steady

state and inflammation, J. Exp. Med., 214, 905-917,

doi:10.1084/jem.20160499.

NEDOSPASOV et al.860

BIOCHEMISTRY (Moscow) Vol. 89 No. 5 2024

33. Kruglov,A., Drutskaya,M., Schlienz,D., Gorshkova,E.,

Kurz,K., Morawietz,L., and Nedospasov,S. (2020) Con-

trasting contributions of TNF from distinct cellular

sources in arthritis, Ann. Rheum. Dis., 79, 1453-1459,

doi:10.1136/annrheumdis-2019-216068.

34. Wen, Y., Rudemiller, N. P., Zhang, J., Robinette, T.,

Lu,X., Ren,J., Privratsky, J.R., Nedospasov, S.A., and

Crowley, S. D. (2020) TNF-α in Tlymphocytes attenu-

ates renal injury and fibrosis during nephrotoxic ne-

phritis, Am.J. Physiol. Renal. Physiol., 318, F107-F116,

doi:10.1152/ajprenal.00347.2019.

35. Dudeck, J., Kotrba, J., Immler, R., Hoffmann, A.,

Voss,M., Alexaki, V.I., Morton,L., Jahn, S.R., Katsoulis-

Dimitriou,K., Winzer,S., Kollias,G., Fischer,T., Nedo-

spasov, S. A., Dunay, I. R., Chavakis, T., Müller, A. J.,

Schraven, B., Sperandio, M., and Dudeck, A. (2021)

Directional mast cell degranulation of tumor necro-

sis factor into blood vessels primes neutrophil ex-

travasation, Immunity, 54, 468-483.e5, doi: 10.1016/

j.immuni.2020.12.017.

36. Ninnemann, J., Winsauer, C., Bondareva, M., Kühl,

A. A., Lozza, L., Durek, P., Lissner, D., Siegmund, B.,

Kaufmann, S. H. E., Mashreghi, M.-F., Nedospasov,

S. A., and Kruglov, A. A. (2022) TNF hampers intesti-

nal tissue repair in colitis by restricting IL-22 bioavail-

ability, Mucosal. Immunol., 15, 698-716, doi: 10.1038/

s41385-022-00506-x.

37. Lakin,R., Polidovitch,N., Yang,S., Parikh,M., Liu, X.,

Debi, R., Gao, X., Chen, W., Guzman, C., Yakobov, S.,

Izaddoustdar, F., Wauchop, M., Lei, Q., Xu, W., Nedo-

spasov, S.A., Christoffels, V.M., and Backx, P.H. (2023)

Cardiomyocyte and endothelial cells play distinct

roles in the tumour necrosis factor (TNF)-dependent

atrial responses and increased atrial fibrillation vul-

nerability induced by endurance exercise training in

mice, Cardiovasc. Res., 119, 2607-2622, doi: 10.1093/

cvr/cvad144.

38. Kroetsch, J.T., Levy, A.S., Zhang,H., Aschar-Sobbi,R.,

Lidington,D., Offermanns,S., Nedospasov, S.A., Backx,

P.H., Heximer, S.P., and Bolz, S.-S. (2017) Constitutive

smooth muscle tumour necrosis factor regulates mi-

crovascular myogenic responsiveness and systemic

blood pressure, Nat. Commun., 8, 14805, doi:10.1038/

ncomms14805.

39. Dinh, D. D., Lidington, D., Kroetsch, J. T., Ng, C.,

Zhang,H., Nedospasov, S.A., Heximer, S.P., and Bolz,

S.-S. (2020) Experimental subarachnoid hemorrhage

drives catecholamine-dependent cardiac and periph-

eral microvascular dysfunction, Front. Physiol., 11,

402, doi:10.3389/fphys.2020.00402.

40. Han,J., Brown,T., and Beutler,B. (1990) Endotoxin-re-

sponsive sequences control cachectin/tumor necrosis

factor biosynthesis at the translational level, J. Exp.

Med., 171, 465-475, doi:10.1084/jem.171.2.465.

41. Yocum, D. E., Esparza, L., Dubry, S., Benjamin, J. B.,

Volz, R., and Scuderi, P. (1989) Characteristics of

tumor necrosis factor production in rheumatoid ar-

thritis, Cell. Immunol., 122, 131-145, doi: 10.1016/

0008-8749(89)90154-8.

42. Keffer, J., Probert, L., Cazlaris, H., Georgopoulos, S.,

Kaslaris, E., Kioussis, D., and Kollias, G. (1991)

Transgenic mice expressing human tumour necro-

sis factor: a predictive genetic model of arthritis,

EMBO J., 10, 4025-4031, doi: 10.1002/j.1460-2075.

1991.tb04978.x.

43. Maini, R.N., Elliott,M., Brennan, F.M., Williams, R.O.,

and Feldmann,M. (1994) Targeting TNF alpha for the

therapy of rheumatoid arthritis, Clin. Exp. Rheumatol.,

12, S63-S66.

44. Feldmann, M., Elliott, M. J., Maini, R. N., and Woody,

J. N. (1997) Anti-tumor necrosis factor-α therapy of

rheumatoid arthritis, Adv. Immunol., 64, 283-350,

doi:10.1016/S0065-2776(08)60891-3.

45. Hayward, M.D., Jones, B. K., Saparov, A., Hain, H.S.,

Trillat, A.-C., Bunzel, M. M., Corona, A., Li-Wang, B.,

Strenkowski,B., Giordano,C., Shen,H., Arcamone,E.,

Weidlick, J., Vilensky, M., Tugusheva, M., Felkner,

R. H., Campbell, W., Rao, Y., Grass, D. S., and

Buiakova, O. (2007) An extensive phenotypic charac-

terization of the hTNFα transgenic mice, BMC Physiol.

7, 13, doi:10.1186/1472-6793-7-13.

46. Lacey, D., Hickey, P., Arhatari, B. D., O’Reilly, L. A.,

Rohrbeck, L., Kiriazis, H., Du, X.-J., and Bouil-

let, P. (2015) Spontaneous retrotransposon inser-

tion into TNF 3′UTR causes heart valve disease and

chronic polyarthritis, Proc. Natl. Acad. Sci. USA, 112,

9698-9703, doi:10.1073/pnas.1508399112.

47. Clayer, E., Dalseno, D., Kueh, A., Lacey, D., Tsai, M.,

Arvell, E. H., Wimmer, V. C., and Bouillet, P. (2020)

Severe impairment of TNF post-transcriptional regu-

lation leads to embryonic death, iScience, 23, 101726,

doi:10.1016/j.isci.2020.101726.

48. Kontoyiannis, D., Pasparakis, M., Pizarro, T. T., Com-

inelli, F., and Kollias, G. (1999) Impaired on/off reg-

ulation of TNF biosynthesis in mice lacking TNF

AU-rich elements: implications for joint and gut-as-

sociated immunopathologies, Immunity, 10, 387-398,

doi:10.1016/s1074-7613(00)80038-2.

49. Liepinsh, D.J., Kruglov, A.A., Galimov, A.R., Shakhov,

A.N., Shebzukhov, Y.V., Kuchmiy, A.A., Grivennikov,

S.I., Tumanov, A.V., Drutskaya, M.S., Feigenbaum,L.,

Kuprash, D.V., and Nedospasov, S.A. (2009) Accelerat-

ed thymic atrophy as a result of elevated homeostatic

expression of the genes encoded by the TNF/lympho-

toxin cytokine locus, Eur. J. Immunol., 39, 2906-2915,

doi:10.1002/eji.200839191.

50. Olleros, M. L., Chavez-Galan, L., Segueni, N.,

Bourigault, M.L., Vesin,D., Kruglov, A.A., Drutskaya,

M. S., Bisig,R., Ehlers, S., Aly, S., Walter, K., Kuprash,

D.V., Chouchkova,M., Kozlov, S.V., Erard,F., Ryffel,B.,

Quesniaux, V. F. J., Nedospasov, S. A., and Garcia, I.

(2015) Control of mycobacterial infections in mice ex-

REVERSE GENETICS APPLIED TO TNF IMMUNOBIOLOGY 861

BIOCHEMISTRY (Moscow) Vol. 89 No. 5 2024

pressing human tumor necrosis factor (TNF) but not

mouse TNF, Infect. Immun., 83, 3612-3623, doi:10.1128/

IAI.00743-15.

51. Ameloot, P., Fiers, W., De Bleser, P., Ware, C. F., Van-

denabeele,P., and Brouckaert,P. (2001) Identification

of tumor necrosis factor (TNF) amino acids crucial for

binding to the murine p75 TNF receptor and construc-

tion of receptor-selective mutants, J.Biol. Chem., 276,

37426-37430, doi:10.1074/jbc.M102020200.

52. Atretkhany, K-S. N., Mufazalov, I. A., Dunst, J., Kuch-

miy, A., Gogoleva, V.S., Andruszewski, D., Drutskaya,

M. S., Faustman, D. L., Schwabenland, M., Prinz, M.,

Kruglov, A. A., Waisman, A., and Nedospasov, S. A.

(2018) Intrinsic TNFR2 signaling in T regulatory

cells provides protection in CNS autoimmunity, Proc.

Natl. Acad. Sci. USA, 115, 13051-13056, doi: 10.1073/

pnas.1807499115.

53. Ruuls, S. R., Hoek, R. M., and Ngo, V.N. (2001) Mem-

brane-bound TNF supports secondary lymphoid organ

structure but is subservient to secreted TNF in driv-

ing autoimmune inflammation, Immunity, 15, 533-543,

doi:10.1016/s1074-7613(01)00215-1.

54. Chudakov, D. M., Matz, M. V., Lukyanov, S., Lukyan-

ov, K. A., Zambito, G., and Mezzanotte, L. (2010) Flu-

orescent proteins and their applications in imaging

living cells and tissues, Physiol. Rev., 90, 1103-1163,

doi:10.1152/physrev.00038.2009.

55. Chawda, C., McMorrow, R., Gaspar, N., Zambito, G.,

and Mezzanotte, L. (2022) Monitoring immune cell

function through optical imaging: a review high-

lighting transgenic mouse models, Mol. Imaging Biol.,

24, 250-263, doi:10.1007/s11307-021-01662-5.

56. Rubtsov, Y.P., Niec, R.E., Josefowicz,S., Li,L., Darce,J.,

Mathis,D., Benoist,C., and Rudensky, A.Y. (2010) Sta-

bility of the regulatory Tcell lineage invivo, Science,

329, 1667-1671, doi:10.1126/science.1191996.

57. Shcherbo,D., Merzlyak, E.M., Chepurnykh, T.V., Frad-

kov, A.F., Ermakova, G.V., Solovieva, E.A., Lukyanov,

K. A., Bogdanova, E.A., Zaraisky, A. G., Lukyanov, S.,

and Chudakov, D.M. (2007) Bright far-red fluorescent

protein for whole-body imaging, Nat. Methods, 4, 741-

746, doi:10.1038/nmeth1083.

58. Shebzukhov, Y. V., Kuchmiy, A. A., Kruglov, A. A.,

Zipp, F., Siffrin, V., and Nedospasov, S. A. (2014) Ex-

perimental applications of TNF-reporter mice with

far-red fluorescent label, in The TNF Superfamily

(Bayry,J., ed.) New York, Springer New York, pp.151-

162, doi:10.1007/978-1-4939-0669-7_13.

59. Kuchmiy, A. A., Kruglov, A. A., Galimov, A. R., Sheb-

zukhov, Yu. V., Chudakov, D.M., Lukyanov, S.A., and

Nedospasov, S. A. (2011) A new line oftransgenic re-

porter mice for the study of expression of the tumor

necrosis factor, Ross. Immunol. Zh., 5, 3-4.

60. Minshawi, F., White, M. R. H., Muller, W., Hum-

phreys,N., Jackson,D., Campbell, B.J., Adamson,A., and

Papoutsopoulou, S. (2019) Human TNF-Luc reporter

mouse: a new model to quantify inflammatory respons-

es, Sci. Rep., 9, 193, doi:10.1038/s41598-018-36969-x.

61. Hall, B. E., Zhang, L., Sun, Z. J., Utreras, E., Prochaz-

kova, M., Cho, A., Terse, A., Arany, P., Dolan, J. C.,

Schmidt, B.L., and Kulkarni, A.B. (2016) Conditional

TNF-α overexpression in the tooth and alveolar bone

results in painful pulpitis and osteitis, J. Dent. Res.,

95, 188-195, doi:10.1177/0022034515612022.

62. Drutskaya, M. S., Efimov, G. A., Astrakhantseva, I. V.,

Kruglov, A. A., and Nedospasov, S. A. (2018) Making

anti-cytokine therapy more selective: studies in mice,

Cytokine, 101, 33-38, doi:10.1016/j.cyto.2016.08.022.

Publisher’s Note. Pleiades Publishing remains

neutral with regard to jurisdictional claims in pub-

lished maps and institutional affiliations.